The AgNPs used here were a gift from C. M. Che in the Department of Chemistry, the University of Hong Kong. This work was funded by the National Natural Science Foundation of China (No. 81600399) and the Science and Technology Project of Guangzhou (No.201707010014).

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
<ol>
	<li>Maintain pregnant BALB/c mice in a specific pathogen-free environment under a 12 h dark/light cycle at 25 &#176;C, with access to autoclaved chow ad libitum.</li>
	<li>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&#39;s modified Eagle&#39;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.
	<ol>
		<li>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 &#176;C with 5% CO2.</li>
		<li>Lyse the infected cells in the culture flask by three freeze-and-thaw cycles, with 20 min in a -80 &#176;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.</li>
		<li>Remove large cellular debris from the lysate by low-speed centrifugation (300 x g at 4 &#176;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 &#176;C or in liquid nitrogen.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Within 24 h of birth, administer to each neonate 20 &#181;L 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.</li>
	<li>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 &#38; 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.</li>
</ol>
2. Silver Nanoparticle Synthesis
<ol>
	<li>Prepare and characterize the AgNPs as previously described12,20. 
	NOTE: The details of preparing and characterizing the AgNPs have been described in publications by C. M. Che&#39;s team at the Department of Chemistry, the University of Hong Kong12,20. 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.</li>
</ol>
3. Preparation of the Silver Nanoparticle Collagen Mixture
NOTE: The AgNP collagen mixture is prepared and characterized as previously described21 and stored at 4 &#176;C. All procedures must be performed on ice.
<ol>
	<li>First, for the collagen preparation, add 490 &#181;L of type I collagen (4 mg/mL) to a 1.5 mL tube and place it on ice.</li>
	<li>Add 100 &#181;L of phosphate-buffered saline (PBS, 10x) to the collagen and mix it with a pipette.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Then, add 10 &#181;L of NaOH (0.2 M) to the above solution.
	<ol>
		<li>To prepare 0.2 M NaOH, add 8 g of NaOH powder to 1 L of distilled water.</li>
	</ol>
	</li>
	<li>Finally, add 400 &#181;L 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 &#176;C; otherwise, it easily solidifies at room temperature.</li>
</ol>
4. Mouse Injection Method
<ol>
	<li>Administer the infected neonatal mice in the treated RRV group with an i.p. injection of 50 &#181;L 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.</li>
	<li>At the beginning of the injection, press the mouse&#39;s leg with the ring finger obliquely over the right thigh, and introduce the needle slowly at a 15&#176; 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.</li>
	<li>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.</li>
	<li>Observe and record the physical appearances of all mice daily, including jaundice and body weight, as well as the survival rate.</li>
</ol>
5. Blood Sample Collection
NOTE: Blood samples of approximately 120 &#181;L are collected by inserting the needle into the heart. After centrifugation, the serum is collected (approximately 70 &#181;L) for liver function testing. The blood collection method is as follows.
<ol>
	<li>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.</li>
	<li>Immobilize the limbs of the mouse and sterilize the upper and lower abdomen with 75% alcohol.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Biochemical Parameter Detection
<ol>
	<li>Use the serum collected in step 5.5 for a biochemical parameter detection.</li>
	<li>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).</li>
</ol>
7. Extrahepatic Cholangiography to Observe the Extrahepatic Bile Duct Patency
NOTE: Perform the entire process under a dissection microscope.
<ol>
	<li>Fully expose the liver, gallbladder, and extrahepatic bile ducts with a cotton swab.</li>
	<li>Observe and photograph the appearance of the liver and bile ducts under a dissection microscope.</li>
	<li>Use ophthalmic forceps to gently hold the bottom of the gallbladder.</li>
	<li>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&#8211;20 &#181;L of methylene blue.</li>
	<li>Observe under a microscope whether the blue color passes through the extrahepatic bile ducts to the jejunum and take a photograph.</li>
</ol>
8. Collection of Fresh Liver Samples for Hematoxylin and Eosin Staining
<ol>
	<li>Fix the fresh mouse liver tissues overnight in 10% formalin.</li>
	<li>Then, embed the fixed liver tissues in paraffin and section them.</li>
	<li>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.</li>
	<li>Finally, observe the histopathology of the liver under a 40X microscope.</li>
</ol>
9. Immunohistochemical Staining of the Hematoxylin and Eosin-stained Tissue Sections
<ol>
	<li>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 &#176;C.</li>
	<li>Remove endogenous peroxidase by exposing the tissue sections for 10 min to a 3% hydrogen peroxide solution.</li>
	<li>Treat the sections with 5% goat serum, to block nonspecific binding.</li>
	<li>Add primary antibodies rabbit-mouse NKG2D (1:100) to the sections, and incubate them overnight at 4 &#176;C.</li>
	<li>Incubate the sections with the appropriate secondary antibodies (HRP-labeled polymer anti-rabbit system) for 30 min at room temperature.</li>
	<li>Visualize the immunohistochemical staining using 3,3&#39;-diaminobenzidine (DAB) as chromogen.</li>
	<li>Observe the sections under a 40X microscope, acquire images, and proceed to analyze them as desired.</li>
</ol>
10. Flow Cytometric Analysis
<ol>
	<li>Gently mince the liver tissue, pass it through a 70 &#181;m cell strainer, and centrifuge it 2x at 270 x g at 4 &#176;C for 4 min.</li>
	<li>Resuspend the cell pellet in RPMI 1640 medium and analyze it by two-color immunofluorescence using monoclonal antibodies.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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P., Jayanthi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia+with+cytomegalovirus.">Biliary atresia with cytomegalovirus.</a> Indian Pediatrics. 49 (2), 157 (2012).</li><li>Mack, C. L., Feldman, A. G., Sokol, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clues+to+the+Etiology+of+Bile+Duct+Injuryin+Biliary+Atresia.">Clues to the Etiology of Bile Duct Injuryin Biliary Atresia.</a> Seminars in Liver Disease. 32, 307-316 (2012).</li><li>Muraji, T., Suskind, D. L., Irie, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia:+a+new+immunological+insight+into+etiopathogenesis.">Biliary atresia: a new immunological insight into etiopathogenesis.</a> Expert Review of Gastroenterology &amp; Hepatology. 3 (6), 599-606 (2009).</li><li>Sokol, R. J., Mack, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiopathogenesis+of+Biliary+Artesia.">Etiopathogenesis of Biliary Artesia.</a> Seminars in Liver Disease. 21, 517-524 (2001).</li><li>Shivakumar, P., Sabla, G. E., Whitington, P., Chougnet, C. A., Bezerra, J. 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The authors have nothing to disclose.

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 effects24,25. A previous study examined the acute toxicity and genotoxicity of AgNPs after a single i.v. injection in a rat exper...

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 BA1,2,3. 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 extrahepatic bile duct obstruction, and finally, liver fibrosis4,5,6.
The commonly used animal model for BA studies involves the inoculation of a neonatal mouse with the rhesus rotavirus (RRV). The mouse typically develops jaundice after 5 - 6 days, showing a low body weight and acholic stools. The role of the immune response in the disease process is critical, especially for natural killer (NK) cells; the depletion of these cells with anti-NKG2D antibody greatly reduces BA-induced damage7. Furthermore, other cells, including CD4+ T cells, CD8+ T cells, dendritic cells, and regulatory T cells, have all been shown to play roles in the disease8,9,10,11. All data suggest the indispensable nature of the immune system in the course of BA.
Silver nanoparticles (AgNPs) have been demonstrated to have beneficial effects against some infectious diseases, including bacterial infections12 and viral infections13,14,15. However, other than dermatological usage, few studies have used AgNPs in a clinical treatment, mostly because of their potential toxicity. In animal experiments, researchers have generally studied the efficacy of AgNPs administered via oral16 or intravenous methods17. However, no other researchers have studied the efficacy of AgNPs administered via an intraperitoneal (i.p.) injection in neonatal mouse experiments, which is a simple and rapid method leading to a more direct effect on the liver and bile ducts while reducing the toxicity to other systems, such as the immune system. AgNPs have been shown to affect NK cell activity18; therefore, we tested the therapeutic effects of AgNPs administered via i.p. injection in the BA mouse model.

<table>
	<tbody>
		<tr>
			<td>BALB/c mouse</td>
			<td>Guangdong Medical Experimental Animal Center</td>
			<td>SYXK2017-0174</td>
			<td>Animal experiment</td>
		</tr>
		<tr>
			<td>Rhesus rotavirus (RRV)</td>
			<td>ATCC</td>
			<td>ATCC&#160;VR-1739</td>
			<td>Establish biliary atresia mouse model</td>
		</tr>
		<tr>
			<td>MA104 cells</td>
			<td>ATCC</td>
			<td>ATCC&#160;CRL-2378.1</td>
			<td>For laboratory use only</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>10569010</td>
			<td>Mammalian Cell Culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher</td>
			<td>10099141</td>
			<td>Mammalian Cell Culture</td>
		</tr>
		<tr>
			<td>collagen Type I</td>
			<td>CORNING</td>
			<td>354236</td>
			<td>For research use only</td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>OXOID</td>
			<td>BR0014G</td>
			<td>For washing</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>1310-73-2</td>
			<td>Adjust the PH value</td>
		</tr>
		<tr>
			<td>AgNP</td>
			<td></td>
			<td></td>
			<td>Antibacterial 
			Note: The AgNps was a gift from Prof CM Che. in the Department of Chemistry, the University of Hong Kong.</td>
		</tr>
		<tr>
			<td>Insulin syringe with integrated needle</td>
			<td>BD</td>
			<td>9161635S</td>
			<td>For medical use</td>
		</tr>
		<tr>
			<td>15-mL Centrifuge Tube</td>
			<td>Corning</td>
			<td>430791</td>
			<td>For laboratory use only</td>
		</tr>
		<tr>
			<td>1.5-mL Microcentrifuge Tube</td>
			<td>GEB</td>
			<td>CT0200-B-N</td>
			<td>For laboratory use only</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE-Ci</td>
			<td>For laboratory use</td>
		</tr>
		<tr>
			<td>Dissecting/Intravital microscope</td>
			<td>Nikon</td>
			<td>SMZ 1000</td>
			<td>For laboratory use</td>
		</tr>
		<tr>
			<td>anti-Mouse NKp46 FITC</td>
			<td>eBioscience</td>
			<td>11-3351</td>
			<td>For research use only</td>
		</tr>
		<tr>
			<td>anti-Mouse CD4 PE-Cyanine5</td>
			<td>eBioscience</td>
			<td>15-0041</td>
			<td>For research use only</td>
		</tr>
		<tr>
			<td>Monoclonal Mouse Anti-Human CD4</td>
			<td>DAKO</td>
			<td>20001673</td>
			<td>For research use only</td>
		</tr>
		<tr>
			<td>anti-NKG2D</td>
			<td>RD</td>
			<td>MAB1547</td>
			<td>For research use only</td>
		</tr>
		<tr>
			<td>BD FACSCanto Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>FACS Canto Plus</td>
			<td>For laboratory use only</td>
		</tr>
	</tbody>
</table>

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.

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...

Watch this Scientific Journal Video about A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice at JoVE.com

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

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 ...

a-silver-nanoparticle-method-for-ameliorating-biliary-atresia

Research

JoVE Journal

Immunology and Infection

7.5K Views.  First Affiliated Hospital of Jinan University. 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.

Video: A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsr&#248;d et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (&#62;95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...