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 border="0" cellpadding="0" cellspacing="0" style="width:640px;" width="640">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Isoflurane</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:143px;">29405</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">5-0 suture</td>
			<td style="width:147px;">Ethicon</td>
			<td>J834G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">7mm Wound Clips</td>
			<td style="width:147px;">Reflex</td>
			<td style="width:143px;">203-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Absorbable gelatin sponge (GelFoam)</td>
			<td style="width:147px;">Pfizer</td>
			<td style="width:143px;">09-0353-01</td>
			<td>Compressed, Any Size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Alcohol Prep Pad</td>
			<td style="width:147px;">VWR</td>
			<td style="width:143px;">75856-902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Animal Clippers</td>
			<td style="width:147px;">Oster</td>
			<td style="width:143px;">78005010002</td>
			<td>Size 40 Blade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Artificial Tears</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:143px;">048272</td>
			<td>Tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Buprenorphine (Buprenex)</td>
			<td>Reckitt Benckiser&#160;</td>
			<td>NDC 12496-0757-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Iodine Prep Pads</td>
			<td style="width:147px;">Nice Pak Products</td>
			<td style="width:143px;">C12400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Sterile Drapes</td>
			<td style="width:147px;">Stoelting</td>
			<td style="width:143px;">50981</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Surgical Gloves</td>
			<td style="width:147px;">Medline</td>
			<td style="width:143px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

incorporation-survivable-liver-biopsy-procedure-mice-to-assess-non

Nanyang Technological University

Research

JoVE Journal

Medicine

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

Use of a Hanging-weight System for Liver Ischemia in Mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Measurement of the Hepatic Venous Pressure Gradient and Transjugular Liver Biopsy

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced chronic liver disease followed by an instruction for transjugular biopsy. Under local anesthesia and ultrasound guidance, a catheter introducer sheath is placed in the right internal jugular vein. Using&#160;fluoroscopic guidance, a balloon catheter is advanced into the inferior vena cava (IVC) and inserted into a large hepatic vein. Correct and sufficient wedge position of the catheter is ensured by injecting contrast media while the balloon is blocking the outflow of the cannulated hepatic vein. After calibrating the external pressure transducer, continuous pressure recordings are obtained with triplicate recordings of the wedged hepatic venous pressure (WHVP) and free hepatic venous pressure (FHVP). The difference between FHVP and WHVP is referred to as HVPG, with values &#8805;10 mm Hg indicating clinically significant portal hypertension (CSPH). Before removing the catheter, pressure readings obtained in the IVC at the same level, as well as the right atrial pressure are recorded.
Finally, a transjugular liver biopsy can be obtained via the same vascular route. Different systems are available; however, core biopsy needles are preferred over aspiration needles, especially for cirrhotic livers. Again, under fluoroscopic guidance a biopsy needle introducer sheath is advanced into an hepatic vein. Next, the transjugular biopsy needle is gently advanced through the introducer sheath: (i) in case of aspiration biopsy, the needle is advanced into the liver parenchyma under aspiration and then removed quickly, or (ii) in case of a core biopsy, the cutting-mechanism is triggered inside the parenchyma. Several separate passages can be safely performed to obtain sufficient liver specimens via transjugular biopsy. In experienced hands, the combination of these procedures takes about 30-45 min.

Here, we present a protocol for measurement of hepatic venous pressure gradient (HVPG),the gold standard to diagnose clinically significant portal hypertension. Moreover, we describe how to perform a transjugular liver biopsy within the same session.

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

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 ...