Veterinary Science Collected Works - Theses
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ItemMorphological and functional studies in a sheep model of pulmonary fibrosis( 2020)Idiopathic pulmonary fibrosis is a lethal progressive respiratory disease with unknown aetiology that occurs predominantly in Western countries. The incidence and prevalence of this disease condition has been shown to be increasing over the years. IPF is localized to the lungs and diagnosed based on the radiologic and histopathological pattern of usual interstitial pneumonia (UIP). Two therapeutic drugs, pirfenidone and nintedanib, have been approved by the FDA in 2014 for treating IPF patients. While these drugs retard the progression of IPF, they do not cure it. Therefore, research is still required to better understand the underlying pathophysiology of IPF, and to develop therapies that halt and/or reverse the fibrosis in the lungs. Animal models are extensively used to elucidate the mechanisms that drive the fibrosis and to trial potential therapies for IPF. While bleomycin mouse models of pulmonary fibrosis are extensively used to investigate the human disease, these models fail to fully replicate the nature of the disease, and often fail in translating the gained knowledge to the clinic. Compared to the human lung, the small size of the murine lung and its dissimilar lung structure and function, can make it difficult to interpret data that is relevant to the human disease. Since many structural and functional aspects of the respiratory systems in large animals are closer to the human respiratory system, our laboratory developed a sheep model of pulmonary fibrosis, using bleomycin as the inducing agent. In this thesis, the sheep model of pulmonary fibrosis was used to study persistent fibrotic and functional changes in lung parenchyma at different stages of the disease progression after inducing fibrosis with bleomycin. A number of drugs were tested in the model, including the FDA approved drug pirfenidone. The effects of Mast cell activity on pulmonary fibrosis was also assessed in this Thesis. In a number of animal models, there are differences in the persistence of fibrosis after bleomycin insult. To determine the persistence of fibrosis in the sheep model, a study was conducted for sixteen weeks to characterise the time course and reversibility of fibrosis that was induced by bleomycin infusion into lung segments. Initially, a segmental approach was used to induce fibrosis with two infusions of bleomycin directed into the appropriate lung segments, two weeks apart. Saline was infused in the contralateral lung lobe to act as an internal healthy-control lung segment. A total of 10 sheep were used in this experiment. Changes in lung function throughout the experiment showed that the lung compliance was significantly poorer in bleomycin-induced lung segment up to eight weeks, and then improved to near normal levels at sixteen weeks after bleomycin. The improvement of lung function was confirmed with histopathological changes at the 16-week timepoint, where the lung parenchyma of bleomycin-infused lung segments exhibited normal tissue architecture. Importantly, results from this study demonstrate that bleomycin-induced fibrosis in sheep lungs resolves during the 8 to 16 week period after the last bleomycin dose. As the FDA approved anti-fibrotic drug, pirfenidone, is now recommended for treating IPF, an experiment was designed to test whether pirfenidone was also efficacious in the sheep model. The experiment was designed with 2 groups of ten sheep, both groups received bleomycin into a caudal lung segment, and saline into a contralateral control lung segment. One sheep group received 2 oral doses of pirfenidone/day for 5 weeks, starting two weeks after the final bleomycin infusion. The control group was treated identically, except that this group received 2 oral doses of vehicle/day. Pirfenidone was able to attenuate both histopathological and physiological readouts of established fibrosis in the sheep model of pulmonary fibrosis. Pirfenidone treatment improved bleomycin-induced fibrotic changes compared to vehicle control, with improved lung compliance, reduced fibrotic changes and collagen content, as well as a reduction in the density of TGFbeta positive cells and myofibroblasts. Overall, this study showed that pirfenidone can attenuate bleomycin-induced lung fibrosis in sheep. Therefore, in future studies with the sheep model, pirfenidone can be used as a benchmark drug to compare the efficacy of other novel therapeutics tested in this model. The role that mast cells play in pulmonary fibrosis is not well understood. Sheep are a good model for studying pulmonary mast cells because the phenotype and density of mast cells in the ovine lung are similar to that observed in the human lung. Two different drugs were used to investigate the role of mast cells in pulmonary fibrosis in sheep. Firstly, senicapoc, a KCa3.1 ion channel blocker which prevents mast cell activation, was compared with the current FDA approved drug, pirfenidone. Both drugs were given orally twice daily for 5 weeks, starting 2 weeks after the final bleomycin infusion. Without any drug treatments, mast cell density was significantly increased in the parenchyma of bleomycin-infused lung segments that were sampled at seven weeks after bleomycin injury. Mast cell density was significantly reduced in bleomycin-infused lung segments after a 5 week treatment with senicapoc. On the other hand, pirfenidone treatment did not attenuate mast cell density in bleomycin-infused sheep lung segments. This was interesting in that while both senicapoc and pirfenidone attenuated fibrosis and improved lung function in this model, only senicapoc retarded the increase in mast cell density that was attributed to bleomycin injury. Secondly, cromolyn sulphate, a mast cell stabilizing drug, was used in the sheep model to investigate whether it could attenuate the fibrosis resulting from bleomycin injury via its effects on mast cells. Cromolyn sulphate, when administered to bleomycin-infused lung segments, did not return either the mast cell density, or histopathological changes, or functional measurements to control levels that are normally observed in the healthy lung. Overall, results presented in this thesis show that the sheep model of pulmonary fibrosis can be used to study underlying pathophysiological mechanisms and treatments relating to IPF. It will be interesting to see whether the knowledge gained from this thesis can be translated to the clinic and contribute to better treatments outcomes.