This work was supported by grants from the National Natural Science Foundation of China (81974056) to R.Z. and from the National Nature Youth Foundation of China (82101808) and Science and Technology Planning Project of Guangzhou (No. 202102020196) to Z.L.

This study was approved by the Institutional Animal Care and Use Committee of Guangzhou Yongnuo Biomedical Animal Center (IACUC-AEWC-F2208020), where all the animal experiments were performed.
1. Establishment of the chronic fibrotic BA mouse model
NOTE: All the animals were kept in a specific pathogen-free (SPF) environment in the same room, and the experiments were conducted in a conventional environment. BALB/c mice on day 12.5 of gestation (age: 10-12 weeks; weight 35-40 g) were kept in a room without specific pathogens at 25&#176;C under a 12 h dark/light cycle and were provided with free access to autoclaved food and water. Neonatal mice, within 24 h after birth (average body weight: 1.5-1.6 g), were selected for the mouse BA model.
<ol>
	<li>Prepare RRV as previously reported16. 
	NOTE: Due to the poor survival status of the model mice, they need to be separated at 21 days old to avoid them being bitten or even killed by other mice in the same cage.</li>
	<li>Divide neonatal mice into three groups: the control group, the RRV group, and the RRV + anti-Ly6G group. Intraperitoneally inject the neonatal mice with 20 &#181;L of RRV (titer: 1.5 x 106 PFU/mL) (RRV group) or saline (control group) within 24 h after birth. To deplete Gr-1+ cells, pretreat each mouse with an intraperitoneal injection of 5 &#181;g of anti-Ly6G antibody 4 h before the RRV injection. 
	NOTE: The anti-Ly6G antibody solution is stored at 2-8 &#176;C and should not be frozen. Take the antibody out before use and put it at room temperature for 30 min to warm up.</li>
	<li>Inject 10 &#181;g of anti-Ly6G antibody into the abdomen of the mouse every 3 days until day 12 after the RRV injection (Figure 1A).</li>
	<li>Check and record the appearance, weight, and survival of all the mice daily.</li>
</ol>
2. Intraperitoneal injection of the mice
<ol>
	<li>Remove newborn mice from the cage. Use a 1 mL insulin syringe to inject 20 &#181;L of RRV solution or 50 &#181;L of anti-Ly6G antibody solution. Pinch the neck skin of the young mouse with the index finger and thumb of one hand, and gently hold the hind legs of the mouse with the ring finger and tail finger to expose the abdomen.</li>
	<li>Lift the needle in an upward incline. Insert the needle into the middle thigh of the right hind leg of the mouse at an angle of 15&#176; with the skin. After moving along the subcutaneous path until the needle reaches the right costal edge of the mouse, direct the needle downward into the abdominal cavity. Then, inject the liquid under the liver of the mouse. 
	NOTE: Newborn mice have their stomach and spleen in the left abdomen. If a needle is inserted on the left side, it is easy to pierce the stomach or&#160;cause splenic hemorrhage.</li>
	<li>Pull the needle out immediately after the injection, and observe for bleeding or leakage at the injection site. If there is any, wipe it with an autoclaved cotton. Return the mouse to its mother&#39;s cage. 
	&#8203;NOTE: In order to reduce the influence of fluid leakage during injection on the experimental results, ensure the injection action is gentle, slowly remove the needle, and press the injection site with a cotton swab for 30 s.</li>
</ol>
3. Collection of sample tissue
<ol>
	<li>On day 12, anesthetize the mice with 4% isoflurane inhalation, and dissect them under a microscope. Insert a 1 mL insulin syringe into the left ventricle of the heart to collect blood. After blood collection, euthanize the mice by inhalation of 4% isoflurane for 10 min. Centrifuge the blood at 400 &#215; g for 5 min at room temperature, and separate the serum for liver function measurements. 
	NOTE: Blood collection must be performed while the mice are alive. If the mice die, the blood will be retained in the blood vessels and can not be collected.</li>
	<li>Photograph the general appearance of the liver and bile duct. Subsequently, dissect the mouse liver and spleen from the surrounding tissues with scissors and tweezers. 
	&#8203;NOTE: The liver and other tissues are collected and reserved at &#8722;80&#176;C for RNA and protein extraction or soaked in 10% formalin for the preparation of histological specimens.</li>
</ol>
4. Fluorescein angiography of the extrahepatic bile duct
<ol>
	<li>After euthanizing the mouse, entirely expose the liver, gallbladder, and extrahepatic bile ducts with scissors and cotton swabs.</li>
	<li>Observe under a posture microscope, inject 5-10 &#181;L of the fluorescent dye rhodamine 123 (20 mg/mL) into the gallbladder with a 1 mL insulin syringe, and take pictures. This process is the same as that reported in a previously16. 
	NOTE: Different mice in the same group are used for the sample tissue collection and fluorescence angiography.</li>
</ol>
5. H&#38;E staining
<ol>
	<li>Immerse fresh mouse liver tissue in 10% formalin for 24 h.</li>
	<li>After embedding the tissue in paraffin, use a paraffin microtome to cut the paraffin block into sections with a thickness of 4 &#181;m, and place two consecutive sections on the same slide. Experienced personnel are required to standardize the operation to avoid finger cuts17.</li>
	<li>Place the slices into a slicing rack, dewax them in xylene, hydrate them successively in absolute ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and distilled water, and soak in each for 5 min.</li>
	<li>Stain the sections with hematoxylin solution for 5 min, and soak them in 1% hydrochloric acid and 75% alcohol for 5 s. Rinse the sections with clean water, and stain with eosin solution for 1 min.</li>
</ol>
6. CK19 and F4/80 immunohistochemical staining
<ol>
	<li>The first three steps are the same as the steps of tissue embedding, sectioning, and dewaxing in the H&#38;E staining section.</li>
	<li>Perform antigen repair with Tris-EDTA buffer (10 mmol/L Tris base, 1 mmol/L EDTA solution, pH 9.0), heat the sections in a microwave oven at 95&#176;C for 10 min, and then remove and cool them naturally to room temperature.</li>
	<li>Place the tissue sections in 3% hydrogen peroxide solution for 10 min to remove endogenous peroxidase.</li>
	<li>Treat the slices with 5% goat serum to block nonspecific binding.</li>
	<li>Add primary rat anti-mouse cytokeratin 19 or rat anti-mouse F4/80 monoclonal antibody to the sections, and incubate overnight at 4 &#176;C.</li>
	<li>Incubate the sections with appropriate secondary antibodies for 30 min at room temperature.</li>
	<li>Use 3,3&#39;-diaminobenzidine (DAB) as a chromogenic agent to observe the chromogenic reaction under the microscope.</li>
	<li>Observe the slices under a 40x microscope to obtain pictures, and analyze them as needed.</li>
</ol>
7. Sirius Red staining
<ol>
	<li>Perform the first three steps of tissue embedding, sectioning, and dewaxing as described in the H&#38;E staining section.</li>
	<li>After the tissue sections are counterstained with hematoxylin, cover each tissue with 50 &#181;L of Sirius Red dye solution at room temperature for 1 h.</li>
	<li>Dry the slides naturally at room temperature for 4 h, add a drop of neutral gum to each slide, and use a coverslip to slowly cover the tissue to avoid bubbles. Leave the slides at room temperature for 24 h to solidify the neutral gum.</li>
	<li>Use a polarized contrast light microscopy to observe the details of collagen deposition; select a clear and suitable field of view, and adjust the brightness and white balance of the microscope&#39;s visual field. Obtain images, and analyze them as needed under a 40x microscope.</li>
</ol>

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The authors have no conflicts of interest to disclose.

In our study, we used anti-Ly6G antibody to eliminate or decrease Gr-1+ cells in a mouse model of biliary atresia infected with RRV to improve acute BA syndrome, prolong the survival rate, and enable BA mice to enter the chronic phase. Reducing the antibody dose can lead to chronic BA with hepatic fibrosis, which indicates that the number of Gr-1+ cells changes the result of BA in the acute and chronic phases. In our previous study, Gr-1+ cells were reduced after the administration of anti-Ly6G antibody, and the overall survival status of the BA mice was improved15. At the same time, it has been reported that Gr-1+ macrophages19, Gr-1+ neutrophils20, Gr-1+ myeloid cells21, and Gr-1+ granulocytes can enhance fibrosis in some animal models22. However, in this study, Gr-1+ cells in mice were partially depleted with low doses of anti-Ly6G antibody, and collagen deposits remained. As a result, more time may be needed to address the disease more thoroughly.
The application of the anti-Ly6G antibody decreased inflammation, partially preserved the bile ducts, and prevented acute BA-induced death in mice. However, only 20% to 30% of the mice entered the chronic phase of BA; this may be related to the time point and the number of injections of anti-Ly6G antibody. We need to further explore this key point to improve the success rate. In addition, we consider that not only Gr-1+ cells but also other immune cells may play important roles, such as NK cells, T cells, B cells, and macrophages.
As for the protocol, some details need to be noted. (i) In order to avoid the leakage of injected drugs, we use a 1 mL insulin syringe with a 0.33 mm diameter needle, because the needle diameter is small, and the needle hole into the syringe is small, which are conducive to reducing the possibility of drug leakage. (ii) Before the injection, the mouse should be fixed to prevent it from moving, avoid drug leakage, and, thus, further ensure the experimental effect. (iii) During drug injection, we injected the drug into the surface or lower margin of the mouse liver as far as possible so that the drug entered the abdomen and fully contacted the liver to take effect. (iv) The injection is usually made from the upper right thigh of the mouse, because the newborn mouse&#39;s stomach and spleen are in the left abdomen, and the stomach is full of milk. If the needle is inserted from the left side, it can easily puncture the stomach, causing milk to flow into the abdomen, or puncture the spleen, causing bleeding.
CD177 is a homolog of Ly6G, and the expression of CD177 in BA patients has been investigated. CD177 can be used as a marker for the early diagnosis of BA in children, indicating that CD177+ cells play a significant role in the course of BA23. By analyzing the RNA sequencing data, our team found that CD177 cells were the dominant population of Gr-1+ cells; meanwhile, Cd177&#8722;/&#8722; BALB/c mice vaccinated with RRV showed a delayed onset of BA and reduced morbidity and mortality18. Thus, our chronic BA mouse model has obvious advantages for studying the pathogenesis and disease progression of BA; it also provides a suitable animal model for studying potential treatments for BA.

Biliary atresia (BA) is a serious hepatobiliary disease that often occurs in infants and young children; specifically, it presents as an obliterative cholangiopathy with neonatal jaundice and pale stools1. Its clinical characteristics are inflammatory destruction of the intrahepatic and extrahepatic bile ducts and progressive fibrosis, which eventually develops into liver failure2. According to statistics, the incidence of BA in Asian countries is higher than in European and American countries, and the incidence of BA in Asian countries is 1/8,000. The etiology of BA includes viral infection, abnormal bile duct development, immune disorders, and genetic variations3. Kasai surgery is the most commonly used method to improve cholestasis in children with BA, but ultimately, this cannot prevent the progression of fibrosis4. The current treatment for BA mainly relies on liver transplantation, which is limited by the lack of liver sources. An in-depth study of the pathogenesis of BA is the most direct means to resolve the challenges of this disease. However, studies on the pathogenesis of BA mainly rely on BA animal models, and it is crucial to select an appropriate animal model.
Most of the histopathological studies of BA have shown that bile duct hyperplasia (BDP), bile thrombosis, and portal vein fibrosis are the most important pathological features of BA, and other pathological features of different grades exist at the same time, such as portal inflammatory cell infiltration and hepatocyte swelling5,6. At present, there are a variety of mouse models that mimic BA, such as the chronic 3,5-die-thoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mouse model with segmental bile duct obstruction and pericholangitis involving the extrahepatic bile ducts7 and the mouse model of liver fibrosis with an intraperitoneal injection of carbon tetrachloride8. The bile duct ligation (BDL) model is characterized by jaundice and rapid portal vein fibrosis9. The alpha-naphthylisothiocyanate (ANIT)-fed mouse model presents with cholangitis confined to the intrahepatic bile duct and hepatocyte injury10. A mouse model with prolonged jaundice is induced by delayed rhesus rotavirus (RRV) inoculation11. Although there are a variety of animal models, especially mouse models, each model has its own limitations. They can only simulate part of the disease characteristics of BA, such as acute inflammation or liver fibrosis in the process of biliary atresia, and there is no model that is highly consistent with the disease process and pathological features of BA.
The classic BA mouse model is induced by RRV, and this model is the most similar model to BA in humans. However, while RRV-induced BA model mice show similar clinical symptoms and pathological characteristics to some of those of extrahepatic biliary atresia, this model lacks liver fibrosis, which greatly limits the in-depth study of the BA mechanisms and the development of new treatments12. Therefore, this study developed a novel BA mouse model of chronic liver fibrosis. Anti-Ly6G antibody was intraperitoneally injected before RRV inoculation on the day of birth. Then, 5-10 &#181;g of anti-Ly6G antibody was injected four times, with gaps of 2 days between each injection. The BA symptoms of mice were improved, the survival time was prolonged, and the mice entered the chronic fibrosis stage. This model not only simulates the acute phase response of BA but also mimics the processes of the liver and progressive fibrosis. Thus, it is a more suitable animal model for the mechanistic study of BA, and it could provide a theoretical basis for developing future treatments for BA.
The Gr-1 molecule is a myeloid-derived cell surface marker originally found to be expressed in neutrophils13. The depletion of anti-Ly6G antibodies reduces circulating neutrophils by more than 90% and alters the response activated by other immune cells. The functions of Gr-1+ cell populations have been reported in various studies using specific scavenging antibodies, with varying effects identified on cytokines and mediated immune protection14. We have studied the function of Gr-1+ cells in RRV-inoculated BA mouse models. However, since the Gr-1 molecule has not been found to be expressed in humans, a similar molecule, CD177, has been studied in BA patients15. Our data prove the significance of Gr-1+ cell populations, particularly in the chronic fibrotic phase of the disease, and provide a suitable animal model to investigate potential BA therapies.

<table><tbody><tr><td>Balb/c mouse</td><td>Guangdong Skarjingda Biotechnology Co., LTD</td><td>20221000112</td><td></td></tr><tr><td>rat anti-mouse Ly6G</td><td>Bio X Cell</td><td>clone 1A8</td><td>West Lebanon, NH</td></tr><tr><td>rat anti-mouse cytokeratin 19</td><td>Developmental Studies Hybridoma Bank</td><td>clone TROMA III</td><td>Iowa City, IA</td></tr><tr><td>rat anti-mouse F4/80</td><td>R&amp;amp;D Systems</td><td>MAB5580</td><td>Minneapolis, MN</td></tr><tr><td>RRV strain&amp;nbsp;</td><td>ATCC</td><td>MMU 18006</td><td>Manassas, VA</td></tr><tr><td>Fluorescent stereomicroscope</td><td>Olympus</td><td>SZX7</td><td></td></tr><tr><td>Leica light microscopy</td><td>Leica Microsystems</td><td>Leica DMI8+DFC7000T</td><td>Wetzlar, Germany</td></tr><tr><td>Hitachi Pre-Analytical Process Automation System</td><td>Hitachi</td><td>7600 Clinical Analyzer</td><td>Tokyo, Japan</td></tr><tr><td>Isoflurane anesthetic</td><td>RWD</td><td>R510-22-10</td><td></td></tr><tr><td>Rhodamine 123</td><td>Sigma-Aldrich</td><td>83702</td><td></td></tr><tr><td>sirius red dye</td><td>Leagene</td><td>DC0041</td><td></td></tr><tr><td>paraffin microtome</td><td>Leica Microsystems</td><td>RM2235</td><td></td></tr><tr><td>neutral gum</td><td>Solarbio</td><td>G8590</td><td></td></tr></tbody></table>

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.

The mice were intraperitoneally injected with 5 &#181;g of anti-Ly6G antibody within 24 h of birth and then intraperitoneally injected with 20 &#181;L of RRV 4 h later. Then, 10 &#181;g of anti-Ly6G antibody was injected every 3 days until day 12 (Figure 1A). The median survival time in the RRV group was 13 days. On the contrary, most of the mice treated with the antibody developed mild jaundice, and no weight loss was observed (Figure 1C). About 20%-30% of the mice had BA syndrome with long-term jaundice and low body weight but survived for more than 42 days. After anti-Ly6G antibody treatment, the number of Gr-1+ cells decreased15, and the mice entered the stage of chronic fibrosis, termed chronic BA. Specimens were collected on day 12 and day 42, and tissue slices stained with Sirius Red showed a gradual increase in liver fibrosis. The chronic BA mice surviving for 42 days were used for detailed analyses. In addition to the small size, the ears, feet, and tail skin showed marked jaundice (blue arrows in Figure 1E). On day 6 after the RRV injection, the feces color of the mice became lighter, and the urine color became darker; this became significantly different from the control group on day 12, when the RRV group mice showed white feces and dark yellow urine. On day 42, the urine and feces of the chronic BA mice also showed obvious features of jaundice (Figure 1D,E). The livers of the BA mice were removed and compared to those of the controls. The livers were smaller (Figure 2B), necrotic lesions were visible to the naked eye (Figure 2A,&#160;black triangle), and a segment of bile duct atresia was also observed extrahepatically (Figure 2A, white asterisks).
Liver tissue section analysis showed that low-dose anti-Ly6G therapy decreased portal vein inflammation. However, inflammatory cell accumulation was still observed in the liver tissue slices at day 42 (Figure 3A). Sirius Red staining showed a small increase in the deposition of collagen in the portal region on day 12 after the RRV injection. Moreover, no significant changes were observed in collagen expression after treatment with the anti-Ly6g antibody, but the collagen expression in BA tissue samples at day 42 was significantly increased (Figure 3B). When examined under a polarized light microscope, it was observed that collagen fibers had accumulated in the adjacent liver tissue. A substantial deposition of collagen, predominantly green, was seen in the BA tissue specimens at day 42 (Figure 3C), and collagen deposition increased further in mice that survived for 42 days without weight gain. With the reduction of portal inflammation, CK19+ bile duct cells were observed on day 12. On day 42, however, mature bile ducts were hardly detectable, even though an increase in CK19+ bile duct cells was seen (Figure 4A,&#160;red arrows indicate bile duct epithelial cells). In the case of extrahepatic bile ducts in mice with chronic BA, serial dissection of the portal region of mice revealed that the extrahepatic bile ducts were blocked and had inflammatory infiltrates of macrophages (Figure 4B, black arrows indicate macrophages).
Compared with RRV alone, treatment with a low dose of anti-Ly6G antibody decreased liver injury in terms of liver enzyme levels on day 12 after RRV inoculation. The alanine aminotransferase and alkaline phosphatase levels in the liver were higher in the chronic BA group than in the normal control group. The most pronounced changes were found in the bilirubin levels, with RRV + anti-Ly6G mice showing reduced TBIL, DBIL, and IBIL levels in acute BA by contrast with RRV alone. In mice with chronic BA, the levels of TBIL, DBIL, and IBIL were increased (Figure 5), indicating that liver function was significantly reduced in the chronic fibrosis stage of BA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65044/65044fig01.jpg" /> 
Figure 1: The effects of anti-Ly6G antibody treatment in a mouse model of biliary atresia infected with RRV. (A) Schematic illustration of low doses of antibodiesto induce acute and chronic phase BA in mice with arrows indicating the time of injection of the antibody and RRV. (B) Survival curves of the mice in the normal control (Cont.) group, rhesus rotavirus (RRV) group, and RRV + anti-Ly6G antibody group. (C) Body weight curves of the mice in each group. (D) Representative images of the mice and their feces and urine in each group on day 12. (E) Representative images of the mice and their feces and urine in each group on day 42. The blue arrows indicate the yellow ears and tail. This figure has been reprinted with permission from Zhang et al.15. <a href="https://www.jove.com/files/ftp_upload/65044/65044fig01large.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/65044/65044fig02.jpg" /> 
Figure 2: The effects of anti-Ly6G antibody treatment on the liver in a mouse model of biliary atresia infected with RRV. (A) Comparison of liver anatomy and extrahepatic bile duct fluorescence angiography between chronic BA mice (right) and normal mice on day 42. (B) Comparison of the liver size between chronic BA mice and normal mice. The black triangle indicates liver necrosis in mice with chronic BA. The white asterisk indicates extrahepatic biliary atresia. This figure has been reprinted with permission from Zhang et al.15. <a href="https://www.jove.com/files/ftp_upload/65044/65044fig02large.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/65044/65044fig03.jpg" /> 
Figure 3: Liver tissue slices of the mice in the normal control (Cont.) group, rhesus rotavirus (RRV) group, and RRV + anti-Ly6G antibody group on day 12 and day 42. (A) Images of liver tissue sections stained with hematoxylin and eosin (H&#38;E). (B) Sirius Red staining showed collagen deposition (PSR). (C) Further observation of the slices using polarized light microscopy (Pol. Light). Abbreviation: PV = portal vein. Scale bar = 50 &#181;m. This figure has been modified from Zhang et al.15. <a href="https://www.jove.com/files/ftp_upload/65044/65044fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65044/65044fig04.jpg" /> 
Figure 4: Immunohistochemical staining of liver tissue slices from the normal control (Cont.) group, rhesus rotavirus (RRV) group, and RRV + anti-Ly6G antibody group on day 12 and day 42. (A) The expression of bile duct epithelial cells marker (CK19) in different treatment groups. (B) Inflammatory cell infiltration of macrophages was demonstrated in different treatment groups. Abbreviation: PV = portal vein. The red arrow indicates bile duct epithelial cells. The black arrow indicates macrophages. Scale bar = 50 &#181;m. This figure has been modified from Zhang et al.15. <a href="https://www.jove.com/files/ftp_upload/65044/65044fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65044/65044fig05.jpg" /> 
Figure 5: Comparison of liver function in mice in different treatment groups. Mouse blood was collected after the experiment and tested in the laboratory department of the hospital. Each group contained three to five mice. *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001. Abbreviations: ALT = alanine aminotransferase; AST = aspartate aminotransferase; ALP = alkaline phosphatase; TBIL = total bilirubin; DBIL = direct bilirubin; IBIL = indirect bilirubin. This figure has been modified from Zhang et al.15. <a href="https://www.jove.com/files/ftp_upload/65044/65044fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia at JoVE.com

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

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 ...

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Research

JoVE Journal

Immunology and Infection

2.3K Views.  South China University of Technology. 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.

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

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

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

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson&#39;s Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

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.

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 ...

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 ...

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. ...

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 ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Biology

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 ...

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 ...