A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

All animal experimental protocols have been approved by the Institutional Animal Care and Use Committee of the Sun Yat-Sen University Laboratory Animal Center (#IACUC-DB-16-0602).

1. Establishing the Biliary Atresia Mouse Model

1. Maintain pregnant BALB/c mice in a specific pathogen-free environment under a 12 h dark/light cycle at 25 °C, with access to autoclaved chow ad libitum.
2. To prepare the RRV strain MMU 18006, amplify the virus in MA104 cells and measure the viral titers by a plaque assay¹⁹.
   NOTE: MA104 cells are cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) in an incubator with a humidified atmosphere containing 5% CO₂. The amplification steps are briefly outlined below.
   1. Infect MA104 cells (1.5 x 10⁷) in a 150 cm² culture flask with trypsin-activated RRV [1.5 x 10⁶ plaque-forming units (PFU)] in 30 mL of serum-free medium. Incubate the infected cells for 3 days in a humidified incubator at 37 °C with 5% CO₂.
   2. Lyse the infected cells in the culture flask by three freeze-and-thaw cycles, with 20 min in a -80 °C freezer for each freeze phase and, then, thawing the cells back to room temperature; the cell-associated virus particle will release into the supernatant. Then, collect and transfer the cell lysate and culture supernatant into a 15 mL conical tube.
   3. Remove large cellular debris from the lysate by low-speed centrifugation (300 x g at 4 °C for 3 min). Then, transfer the supernatant containing the virus (approximately 6 mL) into a new 15 mL conical tube for animal experiments.
      NOTE: The RRV is then ready to be titered, aliquoted, and stored or used for additional rounds of amplification. Long-term exposure to room temperature will reduce the viral infection capacity; the virus should be placed on ice and stored at -80 °C or in liquid nitrogen.
3. Load the RRV into a small-volume (1 mL) insulin syringe with a 29 G needle for the neonatal mouse injection.
   NOTE: The thick needles of volumetric syringes easily lead to drug leakage.
4. Within 24 h of birth, administer to each neonate 20 µL of 1.2 x 10⁵ PFU/mL RRV via the i.p. route; use the same volume of saline as the control.
   NOTE: The syringe used in this experiment is a 1 mL insulin syringe. Infected mice that were not fed by their mothers died within the first 2 days due to other reasons were not included in the analysis.
5. Observe all neonatal mice closely and weigh them daily. Typically, on the sixth day after the RRV inoculation, jaundice appears on the ears and bare skin, the stool becomes clay-colored, and the fur becomes oily, suggesting the establishment of the BA model; check for these symptoms.
   NOTE: BA can be confirmed by a liver tissue section examination with H & E and immunohistochemical stating. The BA mice are then ready for the AgNP treatment.
   CAUTION: The protocol presented is for use with contemporary animal and human RV strains, which must be handled under Biosafety Level 2 (BSL-2) conditions.

2. Silver Nanoparticle Synthesis

1. Prepare and characterize the AgNPs as previously described^12,20.
   NOTE: The details of preparing and characterizing the AgNPs have been described in publications by C. M. Che's team at the Department of Chemistry, the University of Hong Kong^12,20. The final concentration of the solution was 1 mM. The mean diameter of the AgNPs was 10 nm (ranging 5 to 15 nm) and confirmed by electron microscopy.

3. Preparation of the Silver Nanoparticle Collagen Mixture

NOTE: The AgNP collagen mixture is prepared and characterized as previously described²¹ and stored at 4 °C. All procedures must be performed on ice.

1. First, for the collagen preparation, add 490 µL of type I collagen (4 mg/mL) to a 1.5 mL tube and place it on ice.
2. Add 100 µL of phosphate-buffered saline (PBS, 10x) to the collagen and mix it with a pipette.
   1. To prepare 1 L of 10x PBS buffer, combine 80 g of NaCl, 2 g of KCl, and 35.8 g of Na₂HPO₄^.12H₂O to 2.4 g of KH₂PO₄ and store the buffer at room temperature.
3. Then, add 10 µL of NaOH (0.2 M) to the above solution.
   1. To prepare 0.2 M NaOH, add 8 g of NaOH powder to 1 L of distilled water.
4. Finally, add 400 µL of AgNPs (1 mM) to the collagen and mix them with a pipette.
   NOTE: Add the AgNPs last for an even mixing. The AgNP collagen mixture should be stored at 4 °C; otherwise, it easily solidifies at room temperature.

4. Mouse Injection Method

1. Administer the infected neonatal mice in the treated RRV group with an i.p. injection of 50 µL of the AgNP collagen mixture after the appearance of jaundice; perform a second injection 3 d later.
   NOTE: The mice in the control RRV (infected control) group are given the same volume of saline, and the mice in the normal control group are not given any treatment.
2. At the beginning of the injection, press the mouse's leg with the ring finger obliquely over the right thigh, and introduce the needle slowly at a 15° angle (Figure 1). Upon reaching the surface of the lower edge of the liver (Figure 2), about 0.5 cm in, inject the AgNP collagen mixture; then, withdraw the needle slowly.
   NOTE: Be careful not to introduce any air into the syringe as, then, the neonatal mouse may be killed. In neonatal mice, the stomach and spleen are in the left abdomen, and the stomach is full of milk. If the injection is administered from this side, the needle could easily enter either the stomach, causing milk to flow into the abdominal cavity, or the spleen, causing bleeding.
   CAUTION: Pay attention to the needle to prevent any finger injuries, and be sure to replace the needle cap, remove the needle, and place it in a sharps container.
3. After all the injections, keep the mice out of their cages for 10 min to allow the AgNP collagen mixture to gel and to prevent the mother from licking the injection site. Then, return the mice to their cages.
4. Observe and record the physical appearances of all mice daily, including jaundice and body weight, as well as the survival rate.

5. Blood Sample Collection

NOTE: Blood samples of approximately 120 µL are collected by inserting the needle into the heart. After centrifugation, the serum is collected (approximately 70 µL) for liver function testing. The blood collection method is as follows.

1. Anesthetize the mice on the ninth and 12th day after the RRV inoculation (which is 3 days after the AgNP treatment) using 0.5-2.5% sevoflurane.
2. Immobilize the limbs of the mouse and sterilize the upper and lower abdomen with 75% alcohol.
3. Expose the diaphragm by cutting the mouse skin, muscle, and peritoneum along the midline to the xiphoid with scissors; use a sterile cotton swab to remove the gastrointestinal tract to fully expose the diaphragm muscle.
4. Insert the needle (with a 1 mL unloaded insulin syringe) into the left ventricle of the heart and slowly pull back the syringe plunger to obtain the maximum blood volume. Then, transfer the blood to a 1.5 mL tube.
5. Allow the tube to stand for 30 min at room temperature and centrifuge it for 5 min at 400 x g. Then, using a transfer pipette, collect and save the serum for further use.
   NOTE: Avoid damaging the diaphragm, as diaphragm defects easily lead to pneumothorax, death, and blood coagulation, thereby preventing the blood sample collection.

6. Biochemical Parameter Detection

1. Use the serum collected in step 5.5 for a biochemical parameter detection.
2. Use an automated biochemical analyzer to detect the following biochemical parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), globulin (GLO), total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), and total bile acids (TBA).

7. Extrahepatic Cholangiography to Observe the Extrahepatic Bile Duct Patency

NOTE: Perform the entire process under a dissection microscope.

1. Fully expose the liver, gallbladder, and extrahepatic bile ducts with a cotton swab.
2. Observe and photograph the appearance of the liver and bile ducts under a dissection microscope.
3. Use ophthalmic forceps to gently hold the bottom of the gallbladder.
4. Load a 1 mL syringe with methylene blue solution (0.05 wt.% in H₂O). Slowly insert the syringe needle into the gallbladder cavity; then, grasp the needle with the ophthalmic forceps, and slowly infuse 10–20 µL of methylene blue.
5. Observe under a microscope whether the blue color passes through the extrahepatic bile ducts to the jejunum and take a photograph.

8. Collection of Fresh Liver Samples for Hematoxylin and Eosin Staining

1. Fix the fresh mouse liver tissues overnight in 10% formalin.
2. Then, embed the fixed liver tissues in paraffin and section them.
3. Dewax the sections, rehydrate them with an ethanol series (such as 100%, 95%, 80%, and 70% ethanol in distilled water, each for 5 min), stain the tissue sections with hematoxylin, subject them to a 1% hydrochloric acid alcohol differentiation, and finally, stain the sections with eosin.
4. Finally, observe the histopathology of the liver under a 40X microscope.

9. Immunohistochemical Staining of the Hematoxylin and Eosin-stained Tissue Sections

1. After dewaxing and rehydrating the sections, perform an antigen retrieval by submerging the sections in Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA solution; pH 9.0) and heating them in a microwave for 10 min at 95 °C.
2. Remove endogenous peroxidase by exposing the tissue sections for 10 min to a 3% hydrogen peroxide solution.
3. Treat the sections with 5% goat serum, to block nonspecific binding.
4. Add primary antibodies rabbit-mouse NKG2D (1:100) to the sections, and incubate them overnight at 4 °C.
5. Incubate the sections with the appropriate secondary antibodies (HRP-labeled polymer anti-rabbit system) for 30 min at room temperature.
6. Visualize the immunohistochemical staining using 3,3'-diaminobenzidine (DAB) as chromogen.
7. Observe the sections under a 40X microscope, acquire images, and proceed to analyze them as desired.

10. Flow Cytometric Analysis

1. Gently mince the liver tissue, pass it through a 70 µm cell strainer, and centrifuge it 2x at 270 x g at 4 °C for 4 min.
2. Resuspend the cell pellet in RPMI 1640 medium and analyze it by two-color immunofluorescence using monoclonal antibodies.
3. Perform cellular phenotyping using specific cell-surface markers, including fluorescein isothiocyanate- and phycoerythrin-conjugated anti-NKp46 (NK lymphocytes; 1:1,000) and anti-CD4 (T-cell subtype; 1:1,000), with a flow cytometer and analyze the data with flow cytometry data analysis software.
4. Select cell populations according to forward/side scatter, gate according to the isotype controls to account for any background fluorescence, and subject the data to a secondary analysis based on the fluorescence signals from individual antibodies.