The orl rat is more responsive to methacholine challenge than wild type

Abstract

Background: This study presents an animal model of native airway hyperresponsiveness (AHR). AHR is a fundamental aspect of asthma and reflects an abnormal response characterized by airway narrowing following exposure to a wide variety of non-immunological stimuli. Undescended testis (UDT) is one of the most common male congenital anomalies. The orl rat is a Long Evans substrain with inherited UDT. Since boys born with congenital UDT are more likely to manifest asthma symptoms, the main aim of this study was to investigate the alternative hypothesis that orl rats have greater AHR to a methacholine aerosol challenge than wild type rats.

Methods: Long Evans wild type (n = 9) and orl (n = 13) rats were anesthetized, tracheostomized, and mechanically ventilated at 4 weeks of age. Escalating concentrations of inhaled methacholine were delivered. The methacholine potency and efficacy in the strains were measured. Respiratory resistance was the primary endpoint. After the final methacholine aerosol challenge, the short-acting β2-adrenoceptor agonist albuterol was administered as an aerosol and lung/diaphragm tissues were assayed for interleukin (IL)-4, IL-6, and tumor necrosis factor (TNF)-α. Histological and histomorphometrical analyses were performed.

Results: The methacholine concentration-response curve in the orl group indicated increased sensitivity, hyperreactivity, and exaggerated maximal response in comparison with the wild type group, indicating that orl rats had abnormally greater AHR responses to methacholine. Histological findings in orl rats showed the presence of eosinophils, unlike wild type rats. β2-Adrenoceptor agonist intervention resulted in up-regulation of IL-4 diaphragmatic levels and down-regulation of IL-4 and IL-6 in the lungs of orl rats.

Conclusion: orl rats had greater AHR than wild type rats during methacholine challenge, with higher IL-4 levels in diaphragmatic tissue homogenates. Positive immunostaining for IL-4 was detected in lung and diaphragmatic tissue in both strains. This model offers advantages over other pre-clinical murine models for studying potential mechanistic links between cryptorchidism and asthma. This animal model may be useful for further testing of compounds/therapeutics options for treating AHR.

Keywords: Airway hyperresponsiveness; Airway pharmacology; Albuterol sulfate (PubChem CID: 39859); Asthma; Bronchoconstriction; Hyperreactivity; Lung inflammation; Methacholine chloride (PubChem CID: 6114).

Fig. 1

Rat tissue negative controls for IL-4 and mast cell tryptase IHC double-staining. a) Adult rat lung; b) Abdominal wall muscle; c) Pediatric rat lung; d) Diaphragmatic tissue. IL = interleukin; IHC = immuno-histochemistry. Original magnification: 200×.

Fig. 2

Stimulation effects of nebulized methacholine. Schematic representation of the log concentration-response curves observed in wt and orl rats in R. Provocative concentrations (dotted lines) causing a 40% increase in R (PC₄₀) from baseline. Y-axis ordinate shows responses expressed as average % of change from baseline. R = respiratory resistance; wt = wild type. Data are mean ± SEM; ***p < 0.001 for significant interaction in orl (n = 9) vs. wt (n = 13) rats; SEM = standard error of the mean.

Fig. 3

Potency and efficacy of methacholine related to schematic representation of the log concentration-response curves observed in wt (n = 13) and orl rats (n = 9) in Cdyn. Provocative concentrations (dotted lines) causing a 30% decrease in Cdyn (PC₃₀) from baseline. Data are mean ± SEM; Y-axis ordinate shows responses expressed as average % of change from baseline. Cdyn = dynamic compliance; SEM = standard error of the mean; NS = not significant; wt = wild type.

Fig. 4

Percentage of change in R before and after albuterol aerosol treatment. a) wt group (n = 9) and b) orl group (n = 13). †p = 0.057 and **p = 0.009 for comparisons between MCh and albuterol; R = resistance; wt = wild type; MCh = methacholine.

Fig. 5

Levels of cytokines in rat tissue homogenates after MCh aerosol challenge and albuterol aerosol exposure. a) GM differences in lung IL-4 levels. b) Mean ± SEM differences in IL-6 levels. c) Mean ± SEM differences in TNF-α levels, p = 0.207. d) Differences in medians of diaphragmatic IL-4 levels. e) GM differences in IL-6 levels, p = 0.746. f) Differences in medians of TNF-α levels, p = 0.956. IL = interleukin; MCh = methacholine; wt = wild type; TNF-α = tumor necrosis factor-a; GM = geometric mean; *p = <0.05; **p = <0.01 vs. wild type.

Fig. 6

Representative section of nondependent right lung tissues from the Long Evans wild type rats and orl rats. a) Tissue inflammation in wild type rats. b) Tissue inflammation in orl rats. pb = peribronchiolar; pv = perivascular; as = alveolar septal. Hematoxylin and eosin staining; original magnification: 100 .

Fig. 7

Histological scores of pulmonary inflammation in wild type and orl rats. Data are counts of the overall inflammation scores (perivascular, peribronchiolar, and alveolar septal combined). wt = wild type.

Fig. 8

Tissue distribution of IL-4 antigens and mast cell tryptase antigens, as assessed by IHC double-staining analysis in lung and diaphragmatic tissues of rats. a) orl lung; note the thickness of v. b) wt lung. c) orl diaphragm. d) wt diaphragm. IL-4 antigens (brown color/arrows); mast cell antigens (red color/arrow head). Original magnification: 200×. IL = interleukin; IHC = immunohistochemistry; wt = wild type; v = vascular smooth muscle; pb = peribronchiolar; as = alveolar septal; ve = vascular endothelium; as = alveolar septal. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

Levels of cytokines in tissue homogenates of sham-orl rats vs. orl rats. a) GM differences in IL-4 levels. b) Mean ± SEM differences in IL-6 levels. c) Mean ± SEM differences in TNF-α levels. d) GM differences in diaphragmatic IL-4 levels. e) Mean ± SEM differences in IL-6 levels, p = 0.377. f) Differences in medians of TNF-α levels, p = 0.608.