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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;D Systems</td>
			<td>MAB5580</td>
			<td>Minneapolis, MN</td>
		</tr>
		<tr>
			<td>RRV strain&#160;</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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JoVE Journal

Immunology and Infection

2.2K 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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

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 ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...