Computational protocol: Respiratory Syncytial Virus (RSV) Infection in Elderly Mice Results in Altered Antiviral Gene Expression and Enhanced Pathology

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Protocol publication

[…] BALB/c mice were intranasally infected with 105 plaque forming units (pfu)/mouse of either A2, 2-20, or rA2-L19F and euthanized 5 or 8 days post-infection (dpi). Infectious dose was chosen based on references that previously characterize mucogenic strains of RSV in the mouse model , . To compare infection of WT C57BL/6 with OPN-/-, a higher infectious dose (1×106 pfu/mouse) was used because previous reports indicate C57BL/6 are resistant to RSV infections . After euthanasia, chest cavity was opened and the lungs were gently inflated intratracheally with 4°C 4% paraformaldehyde in PBS, removed and immersed in 4% paraformaldehyde at 4°C for an additional 6 hrs. An equal volume of 30% sucrose in PBS was added and tissues were fixed overnight at 4°C. The next day, the solution was replaced with 30% sucrose in PBS and tissues were kept at 4°C for an additional 24 hrs. Fixed tissues were gently blotted before OCT embedding and snap freezing on dry ice. Four 5 µm sections from a single mouse were placed on a single slide and stained with periodic acid-Schiff (PAS) reagent which stains glycoproteins and mucins (ThermoFisher). PAS-stained lungs were viewed with a DP72 digital camera on an IX71Olympus fluorescence microscope; five images were collected from four lengthwise sections per mouse. To quantify cell infiltration, lung images were converted to binary 8-bit formats and maximal points were quantified in areas surrounding bronchioles after noise and background were minimized using the Image-based Tool for Counting Nuclei (ITCN) plug-in with ImageJ. Five bronchioles were quantified per field of view and at least six images were collected from a single mouse. Averages of counts from each mouse (n = 4) were used to generate box plots and analyzed with Minitab software (Minitab Inc., State College, PA) with ANOVA 2-way analysis. PAS-staining was estimated using Color Deconvolution analysis and custom vector settings that were obtained through single-stain vector analysis ( Individual vectors were then used to approximate the area of PAS-stained or hematoxlyin-stained tissue regions, yielding a percentage of PAS/hematoxylin. The complete experiment and histological analysis was performed in duplicate. [...] Mice were intranasally infected with 106 pfu/mouse of the non-mucogenic strain A2 and mice were euthanized at 1, 3 or 5 dpi. The moderate inoculum dose was selected based on multiple references that described intranasal infection with 106 pfu of RSV A2 per mouse is sufficient to induce lung disease and obtain plaque titers from lung homogenates , . Total RNA was isolated from lung tissue and antiviral gene expression was quantitated using RT2-PCR Profiler Arrays (PAMM-122; SABiosciences) in a BioRad CFX96 real-time PCR system. Ct values were obtained using a constant baseline threshold for all PCR runs and samples were run in triplicates using the manufacturer's thermocycler conditions. Five endogenous expression controls provided by the array (Hprt, Hsp90ab1, Gusb, Gapdh, and Actb) were used to calculate the arithmetic mean which was then set as the Ct value for normalization. Scatter-plots were generated using RT2PCR Profiler Data PCR array analysis software and data quality checks were performed. Gene expression changes >2-fold were separately analyzed using GeneGo (Thermo Scientific Inc.) software and GeneMania predictive interaction pathway maps . Biologically relevant networks were assembled using genes associated with >2-fold increase in young mice as compared to aged mice on 1 dpi. Linkages are color coded and indicated co-expression (purple), colocalization (light blue), predicted interactions (orange), shared protein domains (light yellow), or other undefined relationships (gray). Individual gene expression charts were generated to demonstrate the difference in fold-change increases within age groups along a timecourse. Experimental gene expression was defined as the Delta-Delta cycle threshold (ΔΔCt) and calculations were normalized to five endogenous housekeeping genes and in mock-treated, age-matched controls. Heatmaps encompassing the complete 84-gene PCR array were generated using GENE-E software ( Gradients from blue to red indicate minimum to maximum expression of the indicated genes, within each row. Columns indicate the aged and young groups at 1 dpi, 3 dpi, or 5 dpi. One minus the Pearson's correlation value was used for hierarchical clustering; columns indicate timepoint while rows indicate gene examined. Individual mouse RNA array analysis was performed in a single experiment with three different time points (n = 3 mice/group) and gene expression data is displayed as mean values +/- SEM; statistical significance was assessed using 2-way ANOVA. [...] Lung sections were probed for IL-1β and OPN with goat anti-mouse primary antibodies (R&D AF401-NA and AF808, respectively) or goat IgG control antibody then visualized with nickel-3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (ABC Vectastain and DAB kit, Vector Labs); sections were counterstained with hematoxylin and eosin before dehydration, clearing, and mounting of coverslips. To obtain the percentage of hematoxylin-stained cells in the presence of nickel-DAB immunostaining, lung sections were analyzed with ImmunoRatio ImageJ plugin after calibrating to a blank-field correction image and adjusting threshold. Five bronchioles were quantified per field of view and at least 6 images were collected from a single mouse. Percentages from each mouse (n = 4) were used to generate box plots and analyzed with Minitab software with 2-way ANOVA. Prior to collection of images, background and nonspecific nickel-DAB staining was subtracted through comparison of nonspecific goat Ig control antibody. A polyclonal goat antibody for RSV (Millipore, Ab1128) was used to detect RSV antigens in lung sections, followed by indirect immunofluorescence using secondary anti-goat IgG Alexa Fluor-555 conjugated antibody. Sections were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing anti-fade mounting media (SouthernBiotech, Dapi-Fluoromount G) and, after background and blackfield corrections, a minimum of 10 images at 20X magnification were collected per mouse (n = 4) with the DP72 digital camera on an IX71Olympus fluorescence microscope on both fluorescent channels. Single-channel images were used to quantify the number of DAPI-stained nuclei or RSV-positive cells using ITCN ImageJ plugin using differential values for diameters of nuclei versus individual cells, yielding a percentage of RSV-positive cells from total cells per field of view. […]

Pipeline specifications

Software tools ImageJ, Minitab, GeneMANIA, GENE-E, ImmunoRatio
Applications Bright-field microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Infection, Pneumonia