We have successfully used this method to create a mouse model that is more consistent with the pathological changes of the biliary atresia. This technique can simulate the symptoms of acute and chronic biliary atresia which is helpful for future disease mechanism research. After the use of antibody, the survival time of the mice was prolonged and the symptoms were alleviated, suggesting the therapeutic effect of antibody on biliary atresia.
After dividing neonatal mice into different groups, as described in the manuscript, pretreat each mouse with an intraperitoneal injection of five micrograms of anti-Ly6G antibody four hours before the rhesus rotavirus or RRV injection to deplete Gr1-positive cells. Within 24 hours after birth, intraperitoneally inject the neonatal mouse with 20 microliters of rhesus rotavirus or saline. After the RRV injection, administer 20 micrograms of anti-Ly6G antibody into the abdomen of the mouse.
Check and record the appearance, weight, and survival of all the mice daily. For intraperitoneal injection, expose the abdomen of the young mouse by pinching the neck skin with the index finger and thumb of one hand and gently holding the hind legs of the mouse with the ring finger and tail finger. Lift the syringe needle in an upward incline and insert the needle into the middle thigh of the right hind leg of the mouse at a 15-degree angle with the skin.
While moving the needle along the subcutaneous path until it reaches the right costal edge of the mouse, direct the needle down into the abdominal cavity and inject the liquid under the mouse's liver. Pull the needle out immediately after the injection. Observe for bleeding or leakage at the injection site.
After anesthetizing the mouse on the 12th day, dissect it under a microscope. Insert a one-milliliter insulin syringe into the left ventricle of the heart to collect blood. Centrifuge the blood at 400 g for five minutes at room temperature, and separate the serum for liver function measurements.
Dissect the mouse liver and spleen from the surrounding tissues using scissors and tweezers. Photograph the general appearance of the liver and bile duct. After inhalation anesthesia, expose the liver, gallbladder, and extrahepatic bile ducts with scissors and cotton swabs.
Under a posture microscope, inject five to 10 microliters of the fluorescent dye Rhodamine 123 into the gallbladder with a one-milliliter insulin syringe and take pictures. Immerse fresh mouse liver tissue in 10%formalin for 24 hours. Once the tissue is embedded in paraffin, use a paraffin microtome to cut the paraffin block into sections with a thickness of four micrometers, and place two consecutive sections on the same slide.
Then place the slices into a slicing rack, de-wax them in xylene, and hydrate them successively in absolute ethanol, 95%ethanol, 80%ethanol, 70%ethanol, and distilled water for five minutes each. Stain the sections with hematoxylin solution for five minutes, and soak them in 1%hydrochloric acid and 75%alcohol for five seconds. After rinsing the sections with clean water, stain them with eosin solution for one minute.
Prepare the tissue sections for staining, as demonstrated earlier, and perform antigen repair with Tris-EDTA buffer. Heat the sections in a microwave oven at 95 degrees Celsius for 10 minutes, then remove and cool them naturally to room temperature. To remove endogenous peroxidase, place the tissue sections in 3%hydrogen peroxide for 10 minutes, and treat the slices with 5%goat serum to block non-specific binding.
Next, add primary rat anti-mouse Cytokeratin 19 or rat anti-mouse F4/80 monoclonal antibody to the sections, and incubate overnight at four-degree Celsius. Incubate the sections with appropriate secondary antibodies for 30 minutes at room temperature. Use 3, 3-prime-diaminobenzidine as a chromogenic agent to observe the chromogenic reaction under the microscope.
Observe the slices under a 40x microscope to obtain pictures and analyze them as needed. Once the tissue sections are prepared and counterstained with hematoxylin, cover each tissue with 50 microliters of sirius red dye solution at room temperature for one hour. Dry the slides naturally at room temperature for four hours, add a drop of neutral gum to each slide, and use a cover slip to cover the tissue to avoid bubbles.
Leave the slides at room temperature for 24 hours to solidify the neutral gum. Observe the collagen deposition details using polarized contrast light microscopy. Select a clear and suitable field of view, and adjust the brightness and white balance of the microscope's visual field.
Obtain images and analyze them as needed under a 40x microscope. After RRV injection, the median survival time in the RRV group was 13 days. Most of the mice treated with the antibody developed mild jaundice and no weight loss was observed.
The livers of BA mice showed necrotic lesions and an extrahepatic segment of bile duct atresia. The size of the liver is smaller than the controls. Histological analysis showed low-dose anti-Ly6G therapy decreased portal vein inflammation.
Inflammatory cell accumulation was still observed in the liver tissue slices on day 42. On day 12, there was a slight increase in collagen deposition in the portal region. On day 42, the collagen expression was significantly increased, and substantial collagen deposition was seen in the BA tissue.
The CK19-positive cells were observed on day 12. On day 42, CK19-positive cells increased, but there were few mature bile ducts. The extrahepatic bile duct in chronic BA mice were blocked and had inflammatory infiltrates of microphages.
The ALT and ALP levels in the liver were higher in the chronic BA group than in the normal control group. The total bilirubin, direct bilirubin, and indirect bilirubin levels were increased, indicating that liver function was significantly reduced in the chronic fibrosis stage of BA.The injection should be done with caution to not puncture the mouse liver. After the injection, we should take about 10 seconds to observe the state of the mouse and clean the blood from the wound.
