Veterinary Science - Theses
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ItemEpithelial remodelling in a sheep model of asthma( 2013)The airway epithelium is the barrier to inhaled allergens and other agents and thus plays a central role in the restriction of, and response to, such insults. In asthma, ineffective barrier function and aberrant epithelial signalling are believed to be important in both the development and exacerbation of asthma. A review of the literature suggests that rodent models of asthma might not accurately represent the healthy or consequently the diseased airway epithelium of humans. Furthermore, little is known about small airway epithelial remodelling or the long-term effects of allergen exposure in the absence of continued antigenic stimulation. This thesis sought to firstly characterise epithelial remodelling following house dust mite (HDM) allergen challenge in a sheep model of experimental asthma (Chapters 3 and 4). In this model, chronic allergen challenge resulted in the induction of asthmatic features including goblet cell hyperplasia, epithelial hypertrophy, increased EGFR expression and allergen-induced proliferation of airway epithelial cells. Also provided is the first evidence for allergen induced goblet cell degranulation in a non-rodent system, and for degranulation induced via a relevant human allergen (HDM). The long-term effects of allergen exposure on the epithelium were also examined. Epithelial remodelling induced through chronic exposure to HDM remained for three months following the cessation of direct allergen challenge (Chapter 4). Goblet cell hyperplasia, epithelial hypertrophy and epithelial growth factor receptor (EGFR) expression remained in the absence of continued direct allergen challenge, indicative that the epithelium does not require continued allergen exposure to retain an asthmatic phenotype. Microarray analysis of epithelial brushing biopsies (Chapter 5), again demonstrated allergen driven epithelial cell proliferation and a general asthmatic phenotype including the down regulation of cellular tight junction mRNA. Subsequent qPCR validation demonstrated the transcriptional downregulation of the Bone morphogenetic protein (BMP) inhibitor noggin and the tight junction gene occludin in the more severe asthmatic sheep but not in those animals with a less severe phenotype. The downregulation of occludin mRNA was evident both prior to and throughout the establishment of chronic epithelial remodelling. In conclusion this thesis has demonstrated the induction of common asthmatic epithelial changes through chronic allergen challenge in a large animal model, and the retention of this asthmatic phenotype in the absence of continued stimulation. The persistence of epithelial remodelling is an interesting finding given its role as the first line of defence to the external environment. The knowledge gained in this thesis may have useful implications for future therapeutic strategies.
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ItemVascular remodelling and mast cell changes in a sheep model of chronic asthma( 2011)Remodelling of the bronchial vasculature is a recognised disease feature which contributes to the pathogenesis of chronic asthma. Similarly, mast cells are known to play critical roles in allergic airway disease. Research into these areas of asthma pathophysiology can benefit greatly from the use of sheep. This is because the microcirculation, and the distribution and phenotype of mast cells throughout the sheep’s small and large airways are similar to their respective counterparts in human airways. In contrast, the smaller airways in mice do not have an active mast cell component nor do they have an extensive microcirculation, making these species less attractive for studying these aspects of asthma. This thesis uses a sheep model of asthma to characterise the nature and extent of vascular remodelling and changes in mast cell density following repeated exposure to house dust mite (HDM) allergen. Until now, these important airway changes have not been extensively characterised in the sheep model. The thesis aims to correlate these changes with local airway function, and to determine whether they can be inhibited in vivo by administering, a selective inhibitor of the calcium-activated potassium channel KCa3.1, which is known to impede mast cell activation. Initially, a segmental allergen challenge approach was used, whereby sheep sensitised to HDM were given weekly infusions of 1 mg HDM in solution to spatial separate lobes of the lung for different durations. Lung function data shows that repeated HDM allergen exposure results in an increased resting airway resistance, development of bronchial hyperresponsiveness, and acute allergen-induced bronchoconstriction. Morphometric analyses of frozen airway tissue sections revealed that the airways of sheep chronically exposed to HDM allergen exhibit an increased mast cell density and also undergo vascular remodelling. Increases in mast cell density were observed following 16 weeks of repeated allergen exposure, while blood vessel density and vascularity increased with 24 weeks of exposure. Continual allergen exposure was needed to maintain the increased density of mast cells, with numbers decreasing following 12 weeks of allergen withdrawal. Vessel density, however, remained elevated without continual antigenic challenge. There was no correlation between lung function and increased airway vascularity; however, mast cell numbers in HDM-challenged airways correlated with improved airway responses to allergic and non-allergic stimuli. Senicapoc, is an inhibitor of KCa3.1 which has been shown to affect mast cell activation and migration, as well as vascular smooth muscle proliferation. Hence, Senicapoc treatment could potentially prevent vascular remodelling and mast cell-mediated changes in asthma. To further elucidate the role of KCa3.1 in asthma it was ascertained whether Senicapoc administration would influence function, mast cell and vascular remodelling changes in HDM challenged sheep. Twice daily oral administration of Senicapoc for a period of 14 weeks was able to significantly reduce airway responses to allergic and non-allergic stimuli, as well as prevent increases in resting airway resistance following repeated aerosolised challenges of HDM to the whole lung. KCa3.1 blockade did not prevent an increase in airway mast cell density in HDM-challenged sheep; however, it did prevent a significant increase in blood vessel density and resulted in a decrease in mean vessel size. Overall, results presented in this thesis show the usefulness of the sheep model for studying vascular and mast cell changes in small airways, and for examining a therapeutic intervention approach to impede these changes. It will be interesting to see whether intervention strategies derived from this thesis can be applied clinically to treat asthma.