These methods can help answer key questions in biliary atresia. Of BA views about disease, pathogenesis and treatment. The main advantage of this technique is that the neonatal mouse injection technique will help researchers to become familiar with the method for other neonatal mouse model studies.
The implications of this technique extend toward the therapy of BA as several nanoparticles of significance Though this method can provide insight into BA treatment, it can also be applied to other systems such as neonatal mouse models of the pathogenesis of this Generally in the way this to this method will because the small size of neonatal mice can result in building of injection process. To establish biliary atresia in a mouse model, load the rhesus rotovirus into a one milliliter insulin syringe equipped with a 29-gauge needle, and intraperitoneally deliver 1.2 times 10 to the 5th plaque-forming units of virus in 20 microliters of Dulbecco's Modified Eagle Medium to each neonate within 24 hours of birth. Then return the neonates to their mother for daily monitoring and weighing.
When the neonatal animals begin to exhibit signs of jaundice, load a one milliliter syringe equipped with a 29-gauge needle with 50 microliters of freshly prepared silver nanoparticle collagen solution per animal and use the ring finger to press the leg of one jaundiced animal obliquely over the right thigh. Slowly introduce the needle at a 15 degree angle into the peritoneum, and upon reaching the surface of the lower edge of the liver, inject the silver nanoparticle collagen mixture. When all of the mixture has been injected, slowly remove the needle and allow the animal to rest for ten minutes to allow the collagen to solidify and to prevent the mother from licking the injection site.
Then return the mice to their cages for daily monitoring. On day nine or twelve after inoculation, sterilize the upper and lower abdomen with 75%ethanol, and immobilize the limbs of an infected mouse. Open the skin, muscle and peritoneum along the midline to the xyphoid, and use a sterile cotton swab to remove the gastrointestinal tract to fully expose the diaphragm.
Insert the needle of an unloaded one milliliter insulin syringe into the left ventricle of the heart, and slowly retract the syringe plunger to obtain the maximum blood volume. Transfer the blood to a 1.5 milliliter tube for a 30 minute incubation at room temperature and pellet the red blood cells by centrifugation. Then collect the serum for later analysis.
For extrahepatic cholangiography, use a cotton swab to fully expose the liver, gallbladder and extrahepatic bile ducts. After photographing the appearance of the liver and bile ducts under a dissecting microscope, use ophthalmic forceps to gently grasp the bottom of the gallbladder and slowly insert the needle of a one milliliter syringe loaded with methylene blue solution into the gallbladder cavity. Grasping the needle with the forceps, slowly infuse 10-20 microliters of methylene blue.
When the dye passes through the extrahepatic bile ducts to the jejunum, obtain another photograph of the tissue. Then for hematoxylin and eosin staining, harvest the liver from each animal for overnight fixation in 10%formalin. The next morning, embed the samples in paraffin for sectioning, followed by de-waxing re-hydration with an ethanol series and hematoxylin and eosin staining according to standard histopathological analysis protocols.
Compared to untreated biliary atresia mice, silver nanoparticle treated animals demonstrate reduced jaundice and maintain their normal body weight. The levels of bilirubin metabolism and hepatic transaminase drop to normal control values, suggesting that the silver nanoparticles greatly improve liver function. Extrahepatic cholangiography with methylene blue staining confirms bile duct patency after silver nanoparticle treatment, and H&E staining reveals a significant decrease in inflammatory cell infiltration in the hepatic portal area of mice treated with silver nanoparticles compared to controls.
Flow site and metric analysis indicates the presence of significantly fewer NK cells in the livers of silver nanoparticle treated animals compared to untreated rhesus rotavirus infected mice. Further immunohistochemical staining reveals a substantially reduced expression of NK cell marker staining in the portal triad of silver nanoparticle treated mice compared to rhesus rotavirus infected animals. While attempting this procedure, it's important to remember that practice will give best results.
Following this procedure, as methods like conjugating silver nanoparticles to other things can be performed to answer additional questions about the activity of lesions in the treatments of BA in mouse models. is development. This technique provided a way for researchers in the field of gastroenterology to BA or other diseases such as cholestasis in the liver.
Don't forget that working with research rotavirus can be extremely hazardous, and thus precautions such as wearing the appropriate personal protective equipment should always be taken when performing this procedure.
