A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Summary

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.

Abstract

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.

Introduction

BA is a form of cholestasis characterized by persistent jaundice and has high mortality in the absence of liver transplantation. Viral infections are closely associated with the pathogenesis of BA. The cytomegalovirus, reovirus, and rotavirus have all been suggested as pathogens in BA^1,2,3. During the neonatal period, the response of the immature immune system to a viral infection results in immune dysregulation against extra- and intrahepatic bile ducts, leading to biliary epithelial cell apoptosis, inflammatory cell infiltration in the portal area, intrahepatic and extrahep....

Protocol

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 (DM....

Results

Based on the established BA mouse model, the infected neonatal mice were administered an i.p. injection of the prepared AgNP collagen mixture 2x after exhibiting jaundice. Mouse survival was checked for daily, and liver function testing, liver pathology, and flow cytometry were performed. Compared to the untreated control BA mice, the AgNP-treated mice showed reduced jaundice and maintained their normal body weight (Figure 3). The levels of bilirubin metaboli.......

Discussion

AgNPs exhibit potent broad-spectrum antibacterial properties and a strong permeability²²; additionally, they are used to produce a range of antibacterial medical products²³. However, AgNPs can take a long time to clear once they accumulate in organs, and this persistence may lead to toxic effects^24,25. A previous study examined the acute toxicity and genotoxicity of AgNPs after a single i.v. injection in a rat exper.......

Materials

Name	Company	Catalog Number	Comments
BALB/c mouse	Guangdong Medical Experimental Animal Center	SYXK2017-0174	Animal experiment
Rhesus rotavirus (RRV)	ATCC	ATCC VR-1739	Establish biliary atresia mouse model
MA104 cells	ATCC	ATCC CRL-2378.1	For laboratory use only
DMEM	Thermo Fisher	10569010	Mammalian Cell Culture
Fetal Bovine Serum	Thermo Fisher	10099141	Mammalian Cell Culture
collagen Type I	CORNING	354236	For research use only
PBS buffer	OXOID	BR0014G	For washing
NaOH	Sigma	1310-73-2	Adjust the PH value
AgNP			Antibacterial Note: The AgNps was a gift from Prof CM Che. in the Department of Chemistry, the University of Hong Kong.
Insulin syringe with integrated needle	BD	9161635S	For medical use
15-mL Centrifuge Tube	Corning	430791	For laboratory use only
1.5-mL Microcentrifuge Tube	GEB	CT0200-B-N	For laboratory use only
Microscope	Nikon	ECLIPSE-Ci	For laboratory use
Dissecting/Intravital microscope	Nikon	SMZ 1000	For laboratory use
anti-Mouse NKp46 FITC	eBioscience	11-3351	For research use only
anti-Mouse CD4 PE-Cyanine5	eBioscience	15-0041	For research use only
Monoclonal Mouse Anti-Human CD4	DAKO	20001673	For research use only
anti-NKG2D	RD	MAB1547	For research use only
BD FACSCanto Flow Cytometer	BD Biosciences	FACS Canto Plus	For laboratory use only