The authors gratefully acknowledge the assistance of Sally Lee and Holly Koelkebeck for expertise in tissue processing and immunohistochemistry. The authors also thank Donna Goldsteen and the Laboratory Animal Resources staff (MedImmune, Gaithersburg, MD) for their assistance with technical procedures and animal husbandry and care.

All in vivo experiments were conducted in accordance with the policies of the Institutional Animal Care and Use Committee (IACUC) of MedImmune, LLC and in agreement with the Guide for the Care and Use of Laboratory Animals, 8th edition (2011). Male C57BL6J mice were placed onto the high-fat, high-fructose, high-cholesterol AMLN diet. Liver biopsies were performed after 26 weeks on diet, 3 weeks prior to the start of the pharmacological intervention.
1. Obtain and Prepare Surgical Materials
<ol>
	<li>Autoclave sterile instruments (small pointed-tip scissors, blunt tip forceps, cotton swabs, gauze squares, 7 mm staples/wound clips and stapler).</li>
	<li>Obtain, sterile suture for closing abdominal wall (5-0 coated absorbable suture with a PC-1 needle is suitable), absorbable gelatin compressed sponge (to be used for hemostatic purposes), sterile drapes and gloves, iodine-based scrub and 70% ethanol swabs/wipes, buprenorphine (0.05-0.1 mg/kg dose per mouse; at least 3 doses per mouse) and surgical eye ointment/lubrication.</li>
	<li>Prepare vaporizer for anesthesia by filling with isoflurane.</li>
	<li>Turn on a bead sterilizer, for quick instrument sterilization between animals.</li>
	<li>Turn on a warming surface or heating pad, to be used for animal recovery.</li>
	<li>Don appropriate personal protective equipment (PPE) (i.e., mask, gown, hair bonnet, gloves).</li>
</ol>
2. Pre-surgical Preparations
<ol>
	<li>Place the mouse into the induction chamber with 2-3% isoflurane flow to effect.</li>
	<li>Observe the mouse until the lack of response to toe-pinch confirms the depth of anesthesia is appropriate.</li>
	<li>Use an eye ointment to prevent dryness of cornea associated with anesthesia.</li>
	<li>Administer buprenorphine subcutaneously (0.05-0.1 mg/kg).</li>
	<li>Shave the surgical site immediately caudal of the xiphoid process, using animal clippers.</li>
	<li>Move the mouse to a heated surface (37 &#176;C) after shaving the surgical site. Place the mouse in such a way so that the dorsal surface is downward, and the abdomen is exposed. Insert the nose of the mouse into the anesthesia nose cone to facilitate delivery of isoflurane at a flow rate of 2-3%.</li>
	<li>Clean the surgical site with alternating swabs of betadine and 70% ethanol (3 times).</li>
	<li>Open and set up sterile surgical instruments.</li>
	<li>Remove gloves and replace with sterile surgical gloves.</li>
</ol>
3. Surgical Procedure
<ol>
	<li>Make a longitudinal incision with scissors (1-2 cm), immediately caudal of the xiphoid process and slightly to the mouse&#8217;s left side, in the abdominal skin.</li>
	<li>Blunt dissect the skin away from the abdominal wall using forceps and scissors.</li>
	<li>Expose the abdominal wall and visualize liver underneath.</li>
	<li>Using a sterile scalpel blade make an incision (1-2 cm) in the abdominal wall to visualize the left-lateral lobe (LLL) of the liver. 
	NOTE: Be consistent throughout the study. Other common lobes of interest are right-medial lobe (RML) and left-medial lobe (LML).</li>
	<li>Externalize the left lateral lobe through the incision.</li>
	<li>Option 1: Lightly massage the abdomen with the thumb and index finger to expose a portion of the LLL.</li>
	<li>Option 2: Place a sterile cotton swab underneath the LLL to gently pull a portion out to be exposed.</li>
	<li>Place a sterile saline-soaked piece of gauze underneath the LLL to allow a sterile and hydrated surface for the liver to lie on.</li>
	<li>Cut a wedge-shaped piece of the liver from the LLL by making two cuts with sterile scissors. 
	NOTE: The approximate size of the liver being removed is 30-50 mg (&#60;5% of the liver in mice).</li>
	<li>Cut a piece of gelatin sponge similar in size to the wedge piece of liver. Insert the sponge into the cut portion of the liver using forceps.</li>
	<li>Hold the sponge in place with forceps and maintain contact with the liver until it adheres to the surface of the liver and hemostasis is achieved.</li>
	<li>Place the liver biopsy piece into the appropriate tube (i.e. frozen, fixative, etc.).</li>
	<li>Gently place the liver back into the abdominal cavity, taking care to not disturb the sponge.</li>
	<li>Suture the abdominal wall with 5-0 coated absorbable suture starting and ending with a full square knot.</li>
	<li>Close the skin with 7 mm wound clips (or staples).</li>
	<li>Inspect the wound clips to ensure that there is a complete closure of the incision.</li>
	<li>Remove the mouse from the nose cone and place into a clean cage on a heated surface.</li>
	<li>Place instruments into bead sterilizer in between mice for 10-20 s. Make&#160;sure all organic material should be removed from the instruments using sterile saline prior to placing in the glass bead sterilizer.</li>
</ol>
4. Post-operative Care
<ol>
	<li>Monitor the mouse until it regains consciousness and can right itself.</li>
	<li>Record post-operative observations and check the mice daily for dehiscence.</li>
	<li>Administer a second and third dose of buprenorphine (0.05-0.1 mg/kg) subcutaneously 6-8 h post-surgery and again 24 h post-surgery.</li>
	<li>Remove wound clips at 7-10 days post-surgery.</li>
</ol>

<ol>
	<li>Brunt, E. 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=Nonalcoholic+fatty+liver+disease+(NAFLD)+activity+score+and+the+histopathologic+diagnosis+in+NAFLD:+distinct+clinicopathologic+meanings.">Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings.</a> Hepatology. 53 (3), 810-820 (2011).</li><li>Brunt, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+nonalcoholic+fatty+liver+disease.">Pathology of nonalcoholic fatty liver disease.</a> Nature Reviews Gastroenterology and Hepatology. 7 (4), 195-203 (2010).</li><li>Kleiner, D. E., 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=Design+and+validation+of+a+histological+scoring+system+for+nonalcoholic+fatty+liver+disease.">Design and validation of a histological scoring system for nonalcoholic fatty liver disease.</a> Hepatology. 41 (6), 1313-1321 (2005).</li><li>Diehl, A. M., Cause Day, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis,+and+Treatment+of+Nonalcoholic+Steatohepatitis.">Pathogenesis, and Treatment of Nonalcoholic Steatohepatitis.</a> New England Journal of Medicine. 377 (21), 2063-2072 (2017).</li><li>Golabi, 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=Current+complications+and+challenges+in+nonalcoholic+steatohepatitis+screening+and+diagnosis.">Current complications and challenges in nonalcoholic steatohepatitis screening and diagnosis.</a> Expert Reviews of Gastroenterology and Hepatology. 10 (1), 63-71 (2016).</li><li>Roth, N. 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=Prediction+of+histologic+alcoholic+hepatitis+based+on+clinical+presentation+limits+the+need+for+liver+biopsy.">Prediction of histologic alcoholic hepatitis based on clinical presentation limits the need for liver biopsy.</a> Hepatology Communications. 1 (10), 1070-1084 (2017).</li><li>Kristiansen, M. N., 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=Obese+diet-induced+mouse+models+of+nonalcoholic+steatohepatitis-tracking+disease+by+liver+biopsy.">Obese diet-induced mouse models of nonalcoholic steatohepatitis-tracking disease by liver biopsy.</a> World Journal of Hepatology. 8 (16), 673-684 (2016).</li><li>Trevaskis, J. L., 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=Glucagon-like+peptide-1+receptor+agonism+improves+metabolic,+biochemical,+and+histopathological+indices+of+nonalcoholic+steatohepatitis+in+mice.">Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice.</a> American Journal of Physiology Gastrointestinal and Liver Physiology. 302 (8), G762-G772 (2012).</li><li>Tolbol, K. S., 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=Metabolic+and+hepatic+effects+of+liraglutide,+obeticholic+acid+and+elafibranor+in+diet-induced+obese+mouse+models+of+biopsy-confirmed+nonalcoholic+steatohepatitis.">Metabolic and hepatic effects of liraglutide, obeticholic acid and elafibranor in diet-induced obese mouse models of biopsy-confirmed nonalcoholic steatohepatitis.</a> World Journal of Gastroenterology. 24 (2), 179-194 (2018).</li><li>Clapper, J. 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=Diet-induced+mouse+model+of+fatty+liver+disease+and+nonalcoholic+steatohepatitis+reflecting+clinical+disease+progression+and+methods+of+assessment.">Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment.</a> American Journal of Physiology Gastrointestinal and Liver Physiology. 305 (7), G483-G495 (2013).</li><li>Armstrong, M. J., 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=Liraglutide+safety+and+efficacy+in+patients+with+non-alcoholic+steatohepatitis+(LEAN):+a+multicentre,+double-blind,+randomised,+placebo-controlled+phase+2+study.">Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis (LEAN): a multicentre, double-blind, randomised, placebo-controlled phase 2 study.</a> Lancet. 387 (10019), 679-690 (2016).</li></ol>

S.O. and C.R. are current employees and/or stockholders of MedImmune/AstraZeneca. JLT was an employee and stockholder of MedImmune/AstraZeneca at the time these experiments were conducted.

Here we describe in detail the mouse liver biopsy procedure first described by Clapper et&#160;al.10, and exemplify its utility in a mouse model of NASH, similar to the previous reports6,9. The liver biopsy procedure is a valuable tool for assessing NASH phenotype pre- and post-treatment, in a manner similar to that performed in clinical therapeutic trials. The biopsy allows for direct comparison for each subject, i.e. the same mice can be used for both time points. We also demonstrate its translational potential. In a clinical trial in biopsy-proven NASH subjects, liraglutide elicited a 39% response rate (patients with improvement) in NAS, whereas in our study we observed a 66% responder rate for improved NAS. Furthermore, 26% of patients had improved fibrosis score with 9% worsening, versus 14% and 36% in the placebo arm, respectively11. In our study, we observed a 17% improvement rate with liraglutide treatment. These comparisons highlight the power of baseline liver biopsy and confirm potential translational understanding, at least for this class of therapeutic.
It is critical to harvest a large enough piece of liver tissue during the biopsy procedure to properly analyze the histological features, but also not too large of a piece of liver to compromise the function of the liver. We observed a zero-mortality rate with this surgery, and the animals appeared fully recovered within 1 day with the resumption of normal eating behavior. Thus, the time between biopsy and initiation of the intervention study is limited not by animal recovery but likely by the time taken for sample processing, analysis, and subsequent randomization.
Utilizing the liver biopsy procedure as a method of confirmation of fibrosis before intervention helped reduce variability in our study, allowing us to exclude mice that did not fully develop some degree of hepatic fibrosis as a result of the AMLN NASH-inducing diet. Furthermore, it enhanced our ability to better understand the impact of possible NASH-improving interventions, as opposed to only having a terminal liver sample and NASH score. Until new non-invasive techniques are further explored and widely accepted, the liver biopsy is the only proven way to determine NASH.

Non-alcoholic steatohepatitis (NASH) is a progressive liver disease, and a more severe form of nonalcoholic fatty liver disease (NAFLD), that is becoming increasingly prevalent due to its association with obesity and type 2 diabetes. There are no approved therapies for NAFLD/NASH, or approved biomarkers to readily assess the disease progression. NASH is diagnosed histologically by assessing inflammation, excess hepatic lipid (steatosis), hepatocyte ballooning, and fibrosis1,2,3,4. In clinical trials, these endpoints are assessed prior to the study in a liver sample collected via biopsy, and from a second liver biopsy after a certain treatment period. Thus, the effectiveness of the test agent is assessed via change in the liver pathology in subjects with previously identified/quantified (i.e., biopsy-proven) NASH that are randomized to treatment groups. These same histologic endpoints are often used in rodent models assessing potential NASH therapeutics in preclinical studies, but only in terminal tissue from euthanized mice and thus without an understanding of the degree of efficacy at baseline and an inability to control for differences in baseline disease state between treatment groups.
Although 25% of adults with NAFLD are presumed to have NASH based on elevated serum aminotransferase levels, there are no alternative definitive methods to confirm NASH other than histological evidence4. Noninvasive methods, such as radiologic modalities (ultrasound, computed tomography, and magnetic resonance imaging) have been developed to detect hepatic steatosis, but those methods cannot diagnose NASH or determine the stage of fibrosis. Transient elastography offers a promising non-invasive modality for measuring liver stiffness. It has been shown to be accurate in differentiating fibrosis stages. However, the accuracy decreases with higher body mass index (BMI) and fat tissue.5 This is problematic due to the majority of NASH patients being obese. Beyond the clinical relevance of these approaches, none of these modalities are utilized in preclinical studies.
To overcome this, the research group of Dr. Jonathan Roth were the first to implement a liver biopsy procedure in the Amylin NASH (AMLN) mouse model of NASH6,7,8. They described the predictive validity of the biopsy with degree of NASH disease and correlation with metabolic disease. Here we describe in detail the liver biopsy procedure in the AMLN model of NASH and its utility in a therapeutic setting, specifically to assess the efficacy of liraglutide.

<table><tbody><tr><td>Isoflurane</td><td>Henry Schein</td><td>29405</td><td></td></tr><tr><td>5-0 suture</td><td>Ethicon</td><td>J834G</td><td></td></tr><tr><td>7 mm Wound Clips</td><td>Reflex</td><td>203-1000</td><td></td></tr><tr><td>Absorbable gelatin sponge (GelFoam)</td><td>Pfizer</td><td>09-0353-01</td><td>Compressed, Any Size</td></tr><tr><td>Alcohol Prep Pad</td><td>VWR</td><td>75856-902</td><td></td></tr><tr><td>Animal Clippers</td><td>Oster</td><td>78005010002</td><td>Size 40 Blade</td></tr><tr><td>Artificial Tears</td><td>Henry Schein</td><td>048272</td><td>Tube</td></tr><tr><td>Buprenorphine (Buprenex)</td><td>Reckitt Benckiser&amp;nbsp;</td><td>NDC 12496-0757-5</td><td></td></tr><tr><td>Iodine Prep Pads</td><td>Nice Pak Products</td><td>C12400</td><td></td></tr><tr><td>Sterile Drapes</td><td>Stoelting</td><td>50981</td><td></td></tr><tr><td>Surgical Gloves</td><td>Medline</td><td>54720</td><td></td></tr></tbody></table>

incorporation of a survivable liver biopsy procedure in mice to assess non-alcoholic steatohepatitis (nash) resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

For this study, mice were randomized to the treatment groups (n = 12/group) based on pre-study biopsy fibrosis grade and body weight. Mice were administered vehicle (PBS) or liraglutide (5 nmol/kg) for 42 days (vehicle or compound administered 10 mL/kg dose volume, subcutaneously, once daily). On day 42, mice were euthanized (via CO2 inhalation) in the non-fasted state, and liver excised and processed for histology.
Biopsy and terminal liver pieces were fixed overnight in 10% formalin, then paraffin embedded and processed for hematoxylin and eosin, and Sirius Red, staining via standard protocols9. Tissue sections were assessed in a blinded fashion by a pathologist according to the NASH Activity Scoring (NAS) system1,2. The NAS reflects the degree of disease for steatosis, lobular inflammation and hepatocyte ballooning (each parameter given a score of 0-3) as previously described9.
Incorporating a liver biopsy into the design of this study allowed for a baseline (pre-biopsy) fibrosis score. A total of 118 mice were biopsied after 26 weeks of NASH diet and liver fibrosis and steatosis were scored for each biopsy (Figure 1). Of these 118 mice, 49 mice were excluded based on low fibrosis (score of 0), high fibrosis (score of 4, which exhibited abnormal pathology), and abnormal weight loss or wound healing. These exclusion criteria allowed for 69 mice left to assign into study groups and look for improvements in NASH phenotype after treatment (Figure 1).
Mice were treated with vehicle or the GLP-1R agonist liraglutide (5 nmol/kg) for 6 weeks. In the vehicle-treated group, 1 mouse exhibited worsened fibrosis and 4 mice showed worsened NAS score (Figure 2A-B). Liraglutide treatment was associated with an overall improvement of fibrosis (Figure 2C), with 17% of the treatment group improving and 83% remaining unchanged. Similarly, liraglutide treatment improved the overall NAS score (Figure 2D), with 66% of the group having improved NAS score and 33% remained unchanged.
Additionally, liraglutide treatment improved inflammation, with 75% of the group having a lower inflammation score and 25% remained unchanged (Figure 3B). Vehicle treatment worsened inflammation, with 17% of the group having a higher inflammation score and 83% remained unchanged (Figure 3A). Steatosis scoring remained unchanged from baseline to end of the study in both the vehicle- and liraglutide-treated groups (Figure 3A, B). Pre- vs. post-biopsy comparisons for individual mice on inflammation and ballooning parameters are also shown (Figure 3C-F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59130/59130fig1.jpg" /> 
Figure 1: Pre-screening liver biopsy fibrosis and NAS score. (A) Representation of all 118 mice that were screened for fibrosis, via liver biopsy. (B&#160;and C) show the baseline fibrosis and NAS score, respectively, after eligible mice were sorted into study groups. <a href="https://www.jove.com/files/ftp_upload/59130/59130fig1large.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/59130/59130fig2.jpg" /> 
Figure 2: Fibrosis and NAS score, pre-biopsy and post-intervention. Percent responder analysis for the vehicle (A, C) or liraglutide-treated (B, D) mice, as measured from baseline liver biopsy to end of study liver sample for fibrosis stage (A, B) and NAS (C, D). For each group, the change from the pre-study to post-study biopsy is indicated by a line. The points at each scoring step is slightly shifted to allow visual separation of the animals, this is only for visualization purposes and does not reflect any difference in score. <a href="https://www.jove.com/files/ftp_upload/59130/59130fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59130/59130fig3.jpg" /> 
Figure 3: Steatosis, inflammation and ballooning, pre-biopsy and post-intervention. Percent responder analysis for the vehicle (A, C, E) or liraglutide-treated (B, D, F) mice, as measured from baseline liver biopsy to end of study liver sample for steatosis score (A, B), inflammation (C, D) and ballooning (E, F). For each group, the change from the pre-study to post-study biopsy is indicated by a line. The points at each scoring step is slightly shifted to allow visual separation of the animals, this is only for visualization purposes and does not reflect any difference in score. <a href="https://www.jove.com/files/ftp_upload/59130/59130fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution at JoVE.com

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...

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11.8K Views.  MedImmune. In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

Video: Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming increasingly common. Ex vivo liver perfusions allow for a comprehensive analysis of liver metabolism using nuclear magnetic resonance (NMR), in nutritional conditions that can be rigorously controlled. As in silico simulations remain a primarily theoretical means of assessing hormone actions and the effects of pharmaceutical intervention, the perfused liver remains one of the most valuable test beds for understanding hepatic metabolism. As these studies guide basic insights into hepatic physiology, results must be accurate and reproducible. The greatest factor in the reproducibility of ex vivo hepatic perfusion is the quality of surgery. Therefore, we have introduced an organized and streamlined method to perform ex vivo mouse liver perfusions in the context of in situ NMR experiments. We also describe a unique application and discuss common issues encountered in these studies. The overall purpose is to provide an uncomplicated guide to a technique we have refined over several years that we deem the golden standard for obtaining reproducible results in hepatic resections and perfusions in the context of in situ NMR experiments. The distance to the center of the field for the magnet as well as the inaccessibility of the tissue to intervention during the NMR experiment makes our methods novel.

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming ...

Biochemistry

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Immunology and Infection

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 ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

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 ...

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 ...

Impact of Sanleng Jiashen Formula on Orthotopic Hepatocellular Carcinoma in Mice

Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver cancer. Animal models that are repeatable, easy to manipulate, and highly mimic the pathophysiological processes of liver cancer are the prerequisite for the successful screening of effective drug candidates. Meanwhile, reliable drug efficacy evaluation indicators and means are also the guarantee of anti-liver cancer drug research and development.
Sanleng Jiashen formula, a representative prescription of traditional Chinese medicine containing Sparganium stoloni erum, Buch. -Ham. (Sanleng), Panax ginseng C. A. Mey. (ginseng), Rheum officinale Baill. (rhubarb), and Ligusticum chuanxiong Hort. (Chuanxiong), is prescribed to nourish the liver and clear heat, remove toxins, and promote blood circulation to treat liver cancer. This experimental protocol describes the preparation of lyophilized Sanleng Jiashen formula and the establishment process of in-situ liver cancer in BALB/c-nu mice. Histopathological staining, immunohistochemical detection of cancer markers, in vivo imaging of mice, and chick embryo chorioallantoic membrane test were used to explore the inhibition and anti-angiogenesis effect of Sanleng Jiashen formula on malignant proliferation of liver cancer tissue. The data show that the Sanleng Jiashen formula can effectively resist the malignant proliferation of liver cancer tissue, which is manifested by reduced tumor mass volume, improved pathological damage, and lower levels of the cancer marker ki67.
The superior inhibition of angiogenesis also suggests that the Sanleng Jiashen formula may have the potential to treat and prevent the progression and deterioration of liver cancer. The whole experimental scheme shows a comprehensive process of traditional Chinese medicine components in the treatment of mouse liver cancer, which provides a reference for the establishment and optimization of a liver cancer model, as well as the research and development of drugs to prevent and treat liver cancer.

Here, we present the step-by-step protocol to investigate the therapeutic and vascular inhibitory effects of the Sanleng Jiashen formula on BALB/c-nu mice with orthotopic hepatocellular carcinoma.

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

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Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

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

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

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

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

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

Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula