Pulmonary function analysis in cotton rats after respiratory syncytial virus infection

Abstract

The cotton rat (Sigmodon hispidus) is an excellent small animal model for human respiratory viral infections such as human respiratory syncytial virus (RSV) and human metapneumovirus (HMPV). These respiratory viral infections, as well as other pulmonary inflammatory diseases such as asthma, are associated with lung mechanic disturbances. So far, the pathophysiological effects of viral infection and allergy on cotton rat lungs have not been measured, although this information might be an important tool to determine the efficacy of vaccine and drug candidates. To characterize pulmonary function in the cotton rat, we established forced oscillation technique in uninfected, RSV infected and HDM sensitized cotton rats, and characterized pulmonary inflammation, mucus production, pulmonary edema, and oxygenation. There was a gender difference after RSV infection, with females demonstrating airway hyper-responsiveness while males did not. Female cotton rats 2dpi had a mild increase in pulmonary edema (wet: dry weight ratios). At day 4 post infection, female cotton rats demonstrated mild pulmonary inflammation, no increase in mucus production or reduction in oxygenation. Pulmonary function was not significantly impaired after RSV infection. In contrast, cotton rats sensitized to HDM demonstrated airway hyper-responsiveness with a significant increase in pulmonary inflammation, increase in baseline tissue damping, and a decrease in baseline pulmonary compliance. In summary, we established baseline data for forced oscillation technique and other respiratory measures in the cotton rat and used it to analyze respiratory diseases in cotton rats.

Fig 1. Inflammation after RSV A2 infection.

1a. The semi-quantitative scoring of inflammation in lung sections of uninfected cotton rats, cotton rats 5 and 4 days post-RSV infection, and HDM-sensitized cotton rats was compared. The mean and standard deviations are represented (n = 4/ group). Asterisks indicate a p value < 0.05 by One-Way ANOVA. 1b. The total white blood cells per μL of bronchoalveolar lavage fluid (WBC/BALF) were compared between uninfected cotton rats, 5 days post-RSV infected cotton rats, or HDM sensitized cotton rats. Individual points represent the mean WBC/BALF from one animal with the mean and standard deviations represented too; 19DPI AAV-GFP (n = 3), 19DPI AAV-G (n = 3), 5DPI RSV (n = 4). Astericks indicate a p value < 0.05 by One-Way ANOVA. 1c. An H&E stain of an uninfected cotton rat lung representative of no to minimal inflammation. 1d. An H&E stain of a 4dpiRSV cotton rat lung representative of mild inflammation characterized by small numbers of peribronchiolar and perivascular lymphocytes and histiocytes. 1e. An H&E stain of a HDM sensitized cotton rat lung representative of moderate inflammation characterized by many peribronchiolar and perivascular lymphocytes and histiocytes, and numerous granulocytes within alveolar septae (interstitial infiltrates), abundant macrophages and granulocytes within alveoli, and many granulocytes within bronchiolar epithelium.

Fig 2. Mucus production in cotton rats 8 days post-infection with RSV A2 and RSV line 19F.

2a. Quantification of PAS and Alcian blue positive (mucus) staining in bronchioles was performed using the Color Deconvolution algorithm in Image Analysis Toolbox. Mucus production was assessed at 8dpi based on previous studies in the mouse [8, 16]. The mean total percent positive staining cells and standard deviations are represented (n = 5/ group). Asterisks indicate p value < 0.05 when compared to uninfected cotton rats by One-Way ANOVA. 2b. A representative airway and adjacent alveoli of a PAS/ Alcian blue stained section of uninfected cotton rat lung with little to no mucus production. 2c. A representative airway and adjacent alveoli of a PAS/ Alcian blue stained section of cotton rat lungs 8dpi RSV A2 infection with little to no mucus production. 2d. A representative airway and adjacent alveoli of a PAS/ Alcian blue stained section of cotton rat lungs 8dpi RSV line 19F infection with little to no mucus production.

Fig 3. Pulmonary edema after RSV A2 infection.

The lung wet: dry weigh ratios in uninfected cotton rats and cotton rats 2, 4 or 6 days post-RSV infection. Mean wet: dry weight ratios and standard deviations are represented. Asterisks indicate p value < 0.05 when compared to uninfected cotton rats by One-Way ANOVA; uninfected (n = 22), 2DPI RSV (n = 20), 4DPI RSV (n = 20), 6 DPI RSV (n = 8).

Fig 4. Influence of gender on lung mechanics in uninfected and 4 days post-RSV A2 infection.

The mean and standard deviation for each group are represented. Fig a-b represent total airway resistance (R). Fig c-d represent tissue damping (G). Fig e-f represent dynamic pulmonary compliance (C). Fig a, c, and e represent the baseline measurements for R, G, and C. Fig b, d, and f represent the measurements obtained at 50mg/mL methacholine dosage for R, G, and C. Bars indicate p< 0.05 by One-way ANOVA; male uninfected (n = 5), female uninfected (n ranges 7–6), male 4DPI RSV (n ranges 4–3), female 4DPI RSV (n ranges 7–6).

Fig 5. Lung mechanics in uninfected cotton rats and 2, 4, 6 and 28 days post-RSV A2 infection female cotton rats.

The mean and standard deviation for each group are represented. Fig a-b represent total airway resistance (R). Fig c-d represent tissue damping (G). Fig e-f represent dynamic pulmonary compliance (C). Fig a, c, and e represent the baseline measurements for R, G, and C. Fig b, d, and f represent the measurements obtained at 50mg/mL methacholine dosage for R, G, and C. Bars indicate p< 0.05 by One-way ANOVA; uninfected (n ranges 7–6), 2DPI RSV (n = 3), 4DPI RSV (n ranges 7–6), 6DPI RSV (n = 5), 28DPI RSV (n ranges 6–5).

Fig 6. Lung mechanics in uninfected cotton rats and HDM-sensitized female cotton rats.

The mean and standard deviation for each group are represented. Fig a-b represent total airway resistance (R). Fig c-d represent tissue damping (G). Fig e-f represent dynamic pulmonary compliance (C). Fig a, c, and e represent the baseline measurements for R, G, and C. Fig b, d, and f represent the measurements obtained at 50mg/mL methacholine dosage for R, G, and C. Asterisks indicate p< 0.05 by two-tailed unpaired Student T-test; uninfected (n ranges 7–6), HDM (n = 4).