The AgNPs used here were a gift from C. M. Che in the Department of Chemistry, the University of Hong Kong. This work was funded by the National Natural Science Foundation of China (No. 81600399) and the Science and Technology Project of Guangzhou (No.201707010014).

All animal experimental protocols have been approved by the Institutional Animal Care and Use Committee of the Sun Yat-Sen University Laboratory Animal Center (#IACUC-DB-16-0602).
1. Establishing the Biliary Atresia Mouse Model
<ol>
	<li>Maintain pregnant BALB/c mice in a specific pathogen-free environment under a 12 h dark/light cycle at 25 &#176;C, with access to autoclaved chow ad libitum.</li>
	<li>To prepare the RRV strain MMU 18006, amplify the virus in MA104 cells and measure the viral titers by a plaque assay19. 
	NOTE: MA104 cells are cultured in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% fetal bovine serum (FBS) in an incubator with a humidified atmosphere containing 5% CO2. The amplification steps are briefly outlined below.
	<ol>
		<li>Infect MA104 cells (1.5 x 107) in a 150 cm2 culture flask with trypsin-activated RRV [1.5 x 106 plaque-forming units (PFU)] in 30 mL of serum-free medium. Incubate the infected cells for 3 days in a humidified incubator at 37 &#176;C with 5% CO2.</li>
		<li>Lyse the infected cells in the culture flask by three freeze-and-thaw cycles, with 20 min in a -80 &#176;C freezer for each freeze phase and, then, thawing the cells back to room temperature; the cell-associated virus particle will release into the supernatant. Then, collect and transfer the cell lysate and culture supernatant into a 15 mL conical tube.</li>
		<li>Remove large cellular debris from the lysate by low-speed centrifugation (300 x g at 4 &#176;C for 3 min). Then, transfer the supernatant containing the virus (approximately 6 mL) into a new 15 mL conical tube for animal experiments. 
		NOTE: The RRV is then ready to be titered, aliquoted, and stored or used for additional rounds of amplification. Long-term exposure to room temperature will reduce the viral infection capacity; the virus should be placed on ice and stored at -80 &#176;C or in liquid nitrogen.</li>
	</ol>
	</li>
	<li>Load the RRV into a small-volume (1 mL) insulin syringe with a 29 G needle for the neonatal mouse injection. 
	NOTE: The thick needles of volumetric syringes easily lead to drug leakage.</li>
	<li>Within 24 h of birth, administer to each neonate 20 &#181;L of 1.2 x 105 PFU/mL RRV via the i.p. route; use the same volume of saline as the control. 
	NOTE: The syringe used in this experiment is a 1 mL insulin syringe. Infected mice that were not fed by their mothers died within the first 2 days due to other reasons were not included in the analysis.</li>
	<li>Observe all neonatal mice closely and weigh them daily. Typically, on the sixth day after the RRV inoculation, jaundice appears on the ears and bare skin, the stool becomes clay-colored, and the fur becomes oily, suggesting the establishment of the BA model; check for these symptoms. 
	NOTE: BA can be confirmed by a liver tissue section examination with H &#38; E and immunohistochemical stating. The BA mice are then ready for the AgNP treatment. 
	CAUTION: The protocol presented is for use with contemporary animal and human RV strains, which must be handled under Biosafety Level 2 (BSL-2) conditions.</li>
</ol>
2. Silver Nanoparticle Synthesis
<ol>
	<li>Prepare and characterize the AgNPs as previously described12,20. 
	NOTE: The details of preparing and characterizing the AgNPs have been described in publications by C. M. Che&#39;s team at the Department of Chemistry, the University of Hong Kong12,20. The final concentration of the solution was 1 mM. The mean diameter of the AgNPs was 10 nm (ranging 5 to 15 nm) and confirmed by electron microscopy.</li>
</ol>
3. Preparation of the Silver Nanoparticle Collagen Mixture
NOTE: The AgNP collagen mixture is prepared and characterized as previously described21 and stored at 4 &#176;C. All procedures must be performed on ice.
<ol>
	<li>First, for the collagen preparation, add 490 &#181;L of type I collagen (4 mg/mL) to a 1.5 mL tube and place it on ice.</li>
	<li>Add 100 &#181;L of phosphate-buffered saline (PBS, 10x) to the collagen and mix it with a pipette.
	<ol>
		<li>To prepare 1 L of 10x PBS buffer, combine 80 g of NaCl, 2 g of KCl, and 35.8 g of Na2HPO4.12H2O to 2.4 g of KH2PO4 and store the buffer at room temperature.</li>
	</ol>
	</li>
	<li>Then, add 10 &#181;L of NaOH (0.2 M) to the above solution.
	<ol>
		<li>To prepare 0.2 M NaOH, add 8 g of NaOH powder to 1 L of distilled water.</li>
	</ol>
	</li>
	<li>Finally, add 400 &#181;L of AgNPs (1 mM) to the collagen and mix them with a pipette. 
	NOTE: Add the AgNPs last for an even mixing. The AgNP collagen mixture should be stored at 4 &#176;C; otherwise, it easily solidifies at room temperature.</li>
</ol>
4. Mouse Injection Method
<ol>
	<li>Administer the infected neonatal mice in the treated RRV group with an i.p. injection of 50 &#181;L of the AgNP collagen mixture after the appearance of jaundice; perform a second injection 3 d later. 
	NOTE: The mice in the control RRV (infected control) group are given the same volume of saline, and the mice in the normal control group are not given any treatment.</li>
	<li>At the beginning of the injection, press the mouse&#39;s leg with the ring finger obliquely over the right thigh, and introduce the needle slowly at a 15&#176; angle (Figure 1). Upon reaching the surface of the lower edge of the liver (Figure 2), about 0.5 cm in, inject the AgNP collagen mixture; then, withdraw the needle slowly. 
	NOTE: Be careful not to introduce any air into the syringe as, then, the neonatal mouse may be killed. In neonatal mice, the stomach and spleen are in the left abdomen, and the stomach is full of milk. If the injection is administered from this side, the needle could easily enter either the stomach, causing milk to flow into the abdominal cavity, or the spleen, causing bleeding. 
	CAUTION: Pay attention to the needle to prevent any finger injuries, and be sure to replace the needle cap, remove the needle, and place it in a sharps container.</li>
	<li>After all the injections, keep the mice out of their cages for 10 min to allow the AgNP collagen mixture to gel and to prevent the mother from licking the injection site. Then, return the mice to their cages.</li>
	<li>Observe and record the physical appearances of all mice daily, including jaundice and body weight, as well as the survival rate.</li>
</ol>
5. Blood Sample Collection
NOTE: Blood samples of approximately 120 &#181;L are collected by inserting the needle into the heart. After centrifugation, the serum is collected (approximately 70 &#181;L) for liver function testing. The blood collection method is as follows.
<ol>
	<li>Anesthetize the mice on the ninth and 12th day after the RRV inoculation (which is 3 days after the AgNP treatment) using 0.5-2.5% sevoflurane.</li>
	<li>Immobilize the limbs of the mouse and sterilize the upper and lower abdomen with 75% alcohol.</li>
	<li>Expose the diaphragm by cutting the mouse skin, muscle, and peritoneum along the midline to the xiphoid with scissors; use a sterile cotton swab to remove the gastrointestinal tract to fully expose the diaphragm muscle.</li>
	<li>Insert the needle (with a 1 mL unloaded insulin syringe) into the left ventricle of the heart and slowly pull back the syringe plunger to obtain the maximum blood volume. Then, transfer the blood to a 1.5 mL tube.</li>
	<li>Allow the tube to stand for 30 min at room temperature and centrifuge it for 5 min at 400 x g. Then, using a transfer pipette, collect and save the serum for further use. 
	NOTE: Avoid damaging the diaphragm, as diaphragm defects easily lead to pneumothorax, death, and blood coagulation, thereby preventing the blood sample collection.</li>
</ol>
6. Biochemical Parameter Detection
<ol>
	<li>Use the serum collected in step 5.5 for a biochemical parameter detection.</li>
	<li>Use an automated biochemical analyzer to detect the following biochemical parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), globulin (GLO), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), and total bile acids (TBA).</li>
</ol>
7. Extrahepatic Cholangiography to Observe the Extrahepatic Bile Duct Patency
NOTE: Perform the entire process under a dissection microscope.
<ol>
	<li>Fully expose the liver, gallbladder, and extrahepatic bile ducts with a cotton swab.</li>
	<li>Observe and photograph the appearance of the liver and bile ducts under a dissection microscope.</li>
	<li>Use ophthalmic forceps to gently hold the bottom of the gallbladder.</li>
	<li>Load a 1 mL syringe with methylene blue solution (0.05 wt.% in H2O). Slowly insert the syringe needle into the gallbladder cavity; then, grasp the needle with the ophthalmic forceps, and slowly infuse 10&#8211;20 &#181;L of methylene blue.</li>
	<li>Observe under a microscope whether the blue color passes through the extrahepatic bile ducts to the jejunum and take a photograph.</li>
</ol>
8. Collection of Fresh Liver Samples for Hematoxylin and Eosin Staining
<ol>
	<li>Fix the fresh mouse liver tissues overnight in 10% formalin.</li>
	<li>Then, embed the fixed liver tissues in paraffin and section them.</li>
	<li>Dewax the sections, rehydrate them with an ethanol series (such as 100%, 95%, 80%, and 70% ethanol in distilled water, each for 5 min), stain the tissue sections with hematoxylin, subject them to a 1% hydrochloric acid alcohol differentiation, and finally, stain the sections with eosin.</li>
	<li>Finally, observe the histopathology of the liver under a 40X microscope.</li>
</ol>
9. Immunohistochemical Staining of the Hematoxylin and Eosin-stained Tissue Sections
<ol>
	<li>After dewaxing and rehydrating the sections, perform an antigen retrieval by submerging the sections in Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA solution; pH 9.0) and heating them in a microwave for 10 min at 95 &#176;C.</li>
	<li>Remove endogenous peroxidase by exposing the tissue sections for 10 min to a 3% hydrogen peroxide solution.</li>
	<li>Treat the sections with 5% goat serum, to block nonspecific binding.</li>
	<li>Add primary antibodies rabbit-mouse NKG2D (1:100) to the sections, and incubate them overnight at 4 &#176;C.</li>
	<li>Incubate the sections with the appropriate secondary antibodies (HRP-labeled polymer anti-rabbit system) for 30 min at room temperature.</li>
	<li>Visualize the immunohistochemical staining using 3,3&#39;-diaminobenzidine (DAB) as chromogen.</li>
	<li>Observe the sections under a 40X microscope, acquire images, and proceed to analyze them as desired.</li>
</ol>
10. Flow Cytometric Analysis
<ol>
	<li>Gently mince the liver tissue, pass it through a 70 &#181;m cell strainer, and centrifuge it 2x at 270 x g at 4 &#176;C for 4 min.</li>
	<li>Resuspend the cell pellet in RPMI 1640 medium and analyze it by two-color immunofluorescence using monoclonal antibodies.</li>
	<li>Perform cellular phenotyping using specific cell-surface markers, including fluorescein isothiocyanate- and phycoerythrin-conjugated anti-NKp46 (NK lymphocytes; 1:1,000) and anti-CD4 (T-cell subtype; 1:1,000), with a flow cytometer and analyze the data with flow cytometry data analysis software.</li>
	<li>Select cell populations according to forward/side scatter, gate according to the isotype controls to account for any background fluorescence, and subject the data to a secondary analysis based on the fluorescence signals from individual antibodies.</li>
</ol>

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The authors have nothing to disclose.

AgNPs exhibit potent broad-spectrum antibacterial properties and a strong permeability22; additionally, they are used to produce a range of antibacterial medical products23. However, AgNPs can take a long time to clear once they accumulate in organs, and this persistence may lead to toxic effects24,25. A previous study examined the acute toxicity and genotoxicity of AgNPs after a single i.v. injection in a rat exper...

BA is a form of cholestasis characterized by persistent jaundice and has high mortality in the absence of liver transplantation. Viral infections are closely associated with the pathogenesis of BA. The cytomegalovirus, reovirus, and rotavirus have all been suggested as pathogens in BA1,2,3. During the neonatal period, the response of the immature immune system to a viral infection results in immune dysregulation against extra- and intrahepatic bile ducts, leading to biliary epithelial cell apoptosis, inflammatory cell infiltration in the portal area, intrahepatic and extrahepatic bile duct obstruction, and finally, liver fibrosis4,5,6.
The commonly used animal model for BA studies involves the inoculation of a neonatal mouse with the rhesus rotavirus (RRV). The mouse typically develops jaundice after 5 - 6 days, showing a low body weight and acholic stools. The role of the immune response in the disease process is critical, especially for natural killer (NK) cells; the depletion of these cells with anti-NKG2D antibody greatly reduces BA-induced damage7. Furthermore, other cells, including CD4+ T cells, CD8+ T cells, dendritic cells, and regulatory T cells, have all been shown to play roles in the disease8,9,10,11. All data suggest the indispensable nature of the immune system in the course of BA.
Silver nanoparticles (AgNPs) have been demonstrated to have beneficial effects against some infectious diseases, including bacterial infections12 and viral infections13,14,15. However, other than dermatological usage, few studies have used AgNPs in a clinical treatment, mostly because of their potential toxicity. In animal experiments, researchers have generally studied the efficacy of AgNPs administered via oral16 or intravenous methods17. However, no other researchers have studied the efficacy of AgNPs administered via an intraperitoneal (i.p.) injection in neonatal mouse experiments, which is a simple and rapid method leading to a more direct effect on the liver and bile ducts while reducing the toxicity to other systems, such as the immune system. AgNPs have been shown to affect NK cell activity18; therefore, we tested the therapeutic effects of AgNPs administered via i.p. injection in the BA mouse model.

<table><tbody><tr><td>BALB/c mouse</td><td>Guangdong Medical Experimental Animal Center</td><td>SYXK2017-0174</td><td>Animal experiment</td></tr><tr><td>Rhesus rotavirus (RRV)</td><td>ATCC</td><td>ATCC&amp;nbsp;VR-1739</td><td>Establish biliary atresia mouse model</td></tr><tr><td>MA104 cells</td><td>ATCC</td><td>ATCC&amp;nbsp;CRL-2378.1</td><td>For laboratory use only</td></tr><tr><td>DMEM</td><td>Thermo Fisher</td><td>10569010</td><td>Mammalian Cell Culture</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher</td><td>10099141</td><td>Mammalian Cell Culture</td></tr><tr><td>collagen Type I</td><td>CORNING</td><td>354236</td><td>For research use only</td></tr><tr><td>PBS buffer</td><td>OXOID</td><td>BR0014G</td><td>For washing</td></tr><tr><td>NaOH</td><td>Sigma</td><td>1310-73-2</td><td>Adjust the PH value</td></tr><tr><td>AgNP</td><td></td><td></td><td>Antibacterial&lt;br/&gt; Note: The AgNps was a gift from Prof CM Che. in the Department of Chemistry, the University of Hong Kong.</td></tr><tr><td>Insulin syringe with integrated needle</td><td>BD</td><td>9161635S</td><td>For medical use</td></tr><tr><td>15 mL Centrifuge Tube</td><td>Corning</td><td>430791</td><td>For laboratory use only</td></tr><tr><td>1.5 mL Microcentrifuge Tube</td><td>GEB</td><td>CT0200-B-N</td><td>For laboratory use only</td></tr><tr><td>Microscope</td><td>Nikon</td><td>ECLIPSE-Ci</td><td>For laboratory use</td></tr><tr><td>Dissecting/Intravital microscope</td><td>Nikon</td><td>SMZ 1000</td><td>For laboratory use</td></tr><tr><td>anti-Mouse NKp46 FITC</td><td>eBioscience</td><td>11-3351</td><td>For research use only</td></tr><tr><td>anti-Mouse CD4 PE-Cyanine5</td><td>eBioscience</td><td>15-0041</td><td>For research use only</td></tr><tr><td>Monoclonal Mouse Anti-Human CD4</td><td>DAKO</td><td>20001673</td><td>For research use only</td></tr><tr><td>anti-NKG2D</td><td>RD</td><td>MAB1547</td><td>For research use only</td></tr><tr><td>BD FACSCanto Flow Cytometer</td><td>BD Biosciences</td><td>FACS Canto Plus</td><td>For laboratory use only</td></tr></tbody></table>

a silver nanoparticle method for ameliorating biliary atresia syndrome in mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

Based on the established BA mouse model, the infected neonatal mice were administered an i.p. injection of the prepared AgNP collagen mixture 2x after exhibiting jaundice. Mouse survival was checked for daily, and liver function testing, liver pathology, and flow cytometry were performed. Compared to the untreated control BA mice, the AgNP-treated mice showed reduced jaundice and maintained their normal body weight (Figure 3). The levels of bilirubin metaboli...

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A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

a-silver-nanoparticle-method-for-ameliorating-biliary-atresia

Research

JoVE Journal

Immunology and Infection

7.6K Views.  First Affiliated Hospital of Jinan University. Deze methoden kunnen helpen bij het beantwoorden van belangrijke vragen in biliaire atresie. Van BA opvattingen over ziekte, pathogenese en behandeling. Het belangrijkste voordeel van deze techniek is dat de neonatale muis injectie techniek zal onderzoekers helpen om vertrouwd te raken met de methode voor andere neonatale muis model studies.De implicaties van deze techniek uit te breiden tot de therapie van BA als verschillende nanodeeltjes van betekenis Hoewel deze methode inzicht kan...

Video: A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Bioengineering

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

Developmental Biology

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Medicine

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.

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.

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

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 ...

Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the human clinical condition and reproduction of inflammatory events corresponding to the onset of clinical biliary pancreatitis. The severity of pancreatic damage can be assessed by measuring the concentration, speed, and volume of the infused bile acid. This study provides an updated checklist of the materials and methods used in the protocol reproduction and shows the main results from this acute pancreatitis (AP) model. Most of the previous publications have limited themselves to reproducing this model in rats. We have applied this method in mice, which provides additional advantages (i.e., the availability of an arsenal of reagents and antibodies for these animals along with the possibility of working with genetically modified strains of mice) that may be relevant to the study. For acute pancreatitis induction in mice, we present a systematic protocol, with a defined dose of 2.5% sodium taurocholate at an infusion speed 10 &#181;L/min for 3 min in C57BL/6 mice that reaches its maximal level of severity within 12 h of induction and highlight results with outcomes that validate the method. With practice and technique, the total estimated time, from the induction of anesthesia to the completion of the infusion, is 25 min per animal.

Animal models for severe acute pancreatitis enable the study of pathophysiological changes at the initial stage, facilitating observation of the evolution of inflammatory events. Here we provide a protocol for the induction of severe acute biliary pancreatitis by retrograde infusion of sodium taurocholate into the pancreatic duct of anesthetized C57BL/6 mice.

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...

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

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

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

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

Biology

Ameliorating Osteoarthritis in Mice Using Silver Nanoparticles

Knee osteoarthritis (KOA) is one of the most commonly encountered degenerative diseases of the joints in people over 45 years of age. Currently, there are not any effective therapeutics for KOA,and the only end-point strategy is total knee arthroplasty (TKA); therefore, KOA is associated with economic burdens and societal costs. The immune inflammatory response is involved in the occurrence and development of KOA. We previously established a mouse model of KOA using type II collagen. Hyperplasia of the synovial tissue was present in the model, alongside a large number of infiltrated inflammatory cells. Silver nanoparticles have substantial anti-inflammatory effects and have been widely used in tumor therapy and surgical drug delivery. Therefore, we evaluated the therapeutic effects of silver nanoparticles in a collagenase II-induced KOA model. The experimental results showed that silver nanoparticles significantly reduced synovial hyperplasia and the infiltration of neutrophils in the synovial tissue. Hence, this work demonstrates the identification of a novel strategy for OA and provides a theoretical basis for preventing the progress of KOA.

Presented here is a protocol for using silver nanoparticles to effectively ameliorate the acute symptoms of type II collagenase-induced osteoarthritis mice, including synovial inflammation, synovial hyperplasia, vascular hyperplasia, etc.

Knee osteoarthritis (KOA) is one of the most commonly encountered degenerative diseases of the joints in people over 45 years of age. Currently, there are ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

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