Development Of A Guinea Pig Model Of Chronic Asthma With Markedly Thickened Airway Walls: Two Studies Using This Animal Model

Airway remodeling theoretically has the potential to affect airway function, but the in vivo evidence of this is sparse. It is also unclear which measurement would be the indicator for airway remodeling. To assess these questions, we developed a guinea pig model of chronic asthma with markedly thickened airway walls by repeated antigen challenge in sensitized animals. Using this model, we examined (1) whether airway wall thickening really results in an increase in airway responsiveness to nonspecific stimuli, and, if so, which site (large or small airways) contributes most to the increase, and (2) whether the basement membrane thickness in large airways, which are thought to correspond to bronchial biopsy sites in humans, reflects the degree of thickening of entire airway walls.Guinea pigs sensitized by repeated exposure to aerosolized ovalbumin were challenged once a week with inhalation of the same antigen or saline over a 4-, 12- or 24-week period. All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985).Airway responsiveness to inhaled acetylcholine was measured before the first challenge and 5 days after the last challenge by the method previously reported by our laboratory [1]. The animals were then killed, and the lungs were removed and fixed with formalin for histologic examination.Morphometric analysis was performed according to the method described by James et al. [2]. Measurements were made by projecting images onto a computer-assisted graphics tablet. The measurements included the perimeter of the luminal border (Pi) and airway area (Ai), the area enclosed by the inner border of the smooth muscle (Aim) and the area enclosed by the outer border of the smooth muscle (Aem). From the measured dimensions, the entire wall area (Aw) was calculated by subtracting the airway from the area enclosed by the outer border of the smooth muscle (Aw = Aem – Ai). Similarly, the smooth muscle layer area (Asm) was given by (Asm = Aem – Aim) and the mucosal layer area (Amuco) was given by (Amuco = Aim – Ai). To allow comparison of airways of different sizes, data for individual airways were standardized by dividing each area by Air, the theoretical fully dilated internal area (airway) given by Pi2/Aπ. The thickness of the basement membrane was determined in large cartilaginous airways, which are thought to correspond to bronchial biopsy sites in humans, on sections stained with elastica van Gieson’s stain.The airway walls of membranous bronchioles from an animal challenged with aerosolized ovalbumin once a week over 24 weeks were markedly thickened. It was confirmed by immunostaining using anti-human smooth muscle α-actin monoclonal antibody that the area appearing as concentric circles was smooth muscle layer (fig. 1). In the cartilaginous bronchi, only the area of the smooth muscle layer increased significantly compared with controls, whereas in the membranous bronchioles, a significant increase in area was observed in both mucosal and smooth muscle layers (fig. 2). A significant decrease in PC20 acetylcholine compared with the control animals was observed in animals that had received a weekly antigen challenge over 24 weeks but not over 12 weeks. When the PC20 acetylcholine values were related to the airway wall dimensions, the strongest correlation was found with the area of the smooth muscle layer in membranous bronchioles. The thickness of the basement membrane in the animals that had received weekly antigen challenge over 12 or 24 weeks was significantly greater than that of the control animals. When the data obtained from animals challenged over 24 weeks were related to the degree of cartilaginous and membranous airway thickening, the basement membrane thickness in large cartilaginous bronchi correlated weakly with the entire wall area of the same bronchi and correlated strongly with the smooth muscle layer area of the same bronchi and both the smooth muscle layer and the entire airway wall area of membranous bronchioles.We developed animal models that consistently showed airway wall thickening which mimics that of chronic asthmatics, both morphologically and functionally. The studies using this animal model revealed that the thickening of small airway walls rather than large airway walls, particularly the increase in the amount of smooth muscle, is strongly associated with the degree of airway hyperresponsiveness. These findings suggest that the thickening of small airway walls rather than large airway walls plays a more important role in increased airway responsiveness to inhaled acetylcholine, and are consistent with the observation in humans that peripheral airways are an important site for the development of airway hyperresponsiveness in patients with asymptomatic asthma [3].We also examined whether the thickness of the basement membrane, which is the only dimension that is reliably measurable in bronchial biopsy specimens, may reflect the entire airway wall thickening. The basement membrane thickness in large cartilaginous bronchi correlated strongly with the smooth muscle layer area of the same bronchi and both the smooth muscle layer and the entire airway wall area of membranous bronchioles. These findings support the view that measurement of basement membrane thickness in bronchial biopsies is useful for the evaluation of the degree of airway wall thickness in asthma. Further work with this animal model will hopefully produce additional information which may lead to the development of a therapeutic strategy for airway remodeling in asthma.