Veterinary Science Collected Works - Theses
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ItemCharacterisation of the Molecular, and Lung Tissue, Pathogenesis in a Sheep Model of Bleomycininduced Pulmonary Fibrosis( 2021)Idiopathic pulmonary fibrosis (IPF) is one of the most devastating respiratory diseases. It is most frequently diagnosed in older people who are above 55 years of age. It is a progressive fibrotic process in the lungs, in which the mean survival time lies between 3-5 years after diagnosis. The aetiology of IPF is not known, and the disease mechanism is not well understood. The development of animal models for human pulmonary fibrosis are useful for gaining knowledge of physiological and pathological disease mechanisms. They are often used to identify potential therapeutic targets that can be exploited to treat the disease. Establishing a more accurate and representative animal model will provide a more complete understanding of the lung fibrosis and how it relates to the human IPF. Our laboratory has developed a novel bleomycin sheep model for pulmonary fibrosis that shares many of the characteristic features of IPF pathology in humans. The model involves the local bronchoscopic instillation of two doses of bleomycin into specific lung segments of sheep and then assessing the localized bleomycin-induced pathology and segmental lung function. We have examined several aspects of pathology in the model, such as: the structural and functional correlations; the longevity of the pathology; and the microvascular remodelling in the parenchymal fibrosis. It has also been utilized for evaluation of therapeutic effects of some novel drug targets for lung fibrosis. A detailed characterization of the molecular and inflammatory aspects of bleomycin-induced lung fibrosis in sheep has not been studied before. Chapter two of this thesis reports on the histopathology and inflammation components of bleomycin-induced lung fibrosis; in the sheep model and the more frequently used mouse model. I found the unique presence of tertiary lymphoid follicles in bleomycin-treated sheep lung tissue parenchyma which share similar characteristic features of the tertiary lymphoid aggregates described in IPF patients. Importantly, these well- organized tertiary lymphoid follicles were absent in mouse lung parenchyma following bleomycin exposure. I also discovered presence of higher fibrotic fractions and fibrotic scores in bleomycin-injured sheep lungs compared to mouse lungs. In addition, increased infiltration of T and B cells in lungs post bleomycin were examined in sheep. Conversely, the infiltration of these cells declined in the mouse model by 28 days after the first bleomycin dose. In chapter three, I studied endoplasmic reticulum stress (ER stress) and apoptosis in type II alveolar epithelial cells (AECs) and macrophages, which have been identified as key drivers in IPF. The findings showed increased levels of ER- stress and apoptosis in type II AECs and macrophages in sheep lung parenchyma after bleomycin damage. Moreover, the elevated levels of ER stress and apoptosis are alleviated by in vivo blockade of the KCa3.1 ion channel in the sheep model for pulmonary fibrosis. In chapter four, I studied the molecular pathogenesis of the disease by evaluating phylogenetically conserved small non-coding RNA (miRNA) expression profiles in the sheep model of lung fibrosis. I found that 49 miRNAs were significantly expressed in sheep lung tissues due to bleomycin injury, which includes the most common miRNA families that are closely related to IPF. Importantly, 18 miRNAs were significantly expressed only in the sheep model and IPF patients, while 17 miRNAs were commonly expressed in sheep and mouse models, and IPF patients, that are yet to be studied. In addition, pathway enrichment analysis of 49 differentially expressed miRNAs showed a significant enrichment of the pathways that are closely associated with the IPF pathogenesis. Overall, the sheep model presented in this thesis potentially serves as a valuable bridging model for lung fibrosis, as it will contribute to the increased translational value of animal trials to predict if treatments can be successful in clinical settings. While I have found many exciting findings with respect to molecular and pathological alterations that occur in injured sheep lungs, and potential drug treatments that can be exploited to ameliorate the disease, there is still much to learn about this disease and its potential therapeutic treatments. The insights of this thesis create exciting new avenues for basic and clinical research.
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ItemMicrovascular remodelling in a sheep model of bleomycin-induced pulmonary fibrosis( 2020)Idiopathic pulmonary fibrosis (IPF) is a lethal, progressive, and chronic lung disease with unknown cause and dismal prognosis. The median survival time of IPF patients is 2-4 years after diagnosis and the treatment options available for IPF are very limited. The pathogenesis of IPF is complex and not fully understood. The current paradigms focus on alveolar epithelial cell injury as a key initiating event followed by dysregulated wound healing process resulting in fibrosis and distortion of the lung’s architecture. During this process, injuries to the lung are not only associated with fibrosis of the lung parenchyma, but also with changes to the pulmonary vasculature, leading to an aberrant vascular remodelling. However, the precise relationship between vascular remodelling, and the development of fibrosis, in the lung parenchyma is not well understood. One of the reasons for this is that the underlying mechanisms that operate in these disease processes are largely unexplored. Initially, using tissues from previously published studies, I confirmed the presence of microvascular remodelling in small airways as well as in the parenchyma of bleomycin-infused fibrotic lung segments. Blood vessel density, particularly those of blood vessels >30um in diameter, was significantly lower in small airways. The small airways that were exposed to bleomycin injury had increased levels of connective tissues in the airway walls which led to functional changes of increased resistance in these lung segments. In contrast to the lower blood vessel density levels found in bleomycin exposed airways, there was increased neovascularisation in the parenchyma of fibrotic lung segments injured by bleomycin. The blood vessel density in the parenchyma correlated with the level of fibrosis in bleomycin infused lung segments. After confirming the microvascular remodelling in response to bleomycin, the effects of different anti-fibrotic and anti-angiogenic treatments on this process were investigated. I found that these microvascular remodelling changes were significantly attenuated by treatment with the KCa3.1 channel inhibitor, senicapoc. The microvascular remodelling was associated with increases in vascular endothelial growth factor (VEGF) expression and endothelial cell proliferation which were significantly reduced with the senicapoc treatment. These parameters were not significantly suppressed with pirfenidone treatment. Senicapoc treatment attenuated microvascular remodelling through inhibition of capillary endothelial cell proliferation and VEGF expression. I further assessed whether blocking of angiogenesis, using a known antiangiogenic agent tetrathiomolybdate, can attenuate bleomycin-induced fibrosis in the sheep model. In this experiment, I used the segmental approach, where two bronchoscopic boluses of bleomycin, administered two weeks apart, were infused into lower lung segments to induce fibrosis. Saline was infused into the contralateral lung segment to act as an internal healthy-control lung segment. Tetrathiomolybdate treatment, via intravenous injection, was started one week after the final bleomycin infusion and continued twice weekly for six weeks. The sheep were then culled, and a full histopathological and other analyses were performed on the extracted lung tissues. The functional parameter of compliance was measured via a wedged bronchoscope procedure prior to and seven weeks following final bleomycin infusion. Bronchoalveolar lavage (BAL) fluid was also assessed sequentially during the study periods. I found that copper lowering by tetrathiomolybdate chelation, results in the attenuation of angiogenesis and fibrosis in lung segments injured by bleomycin. Moreover, tetrathiomolybdate treatment attenuated the histopathological changes, and improved the poorer lung function that were associated with the development of bleomycin-induced pulmonary fibrosis. Tetrathiomolybdate also prevented accumulation of inflammatory cells in BAL fluid two weeks after bleomycin injury. Overall, the results presented in this thesis indicate the presence of microvascular remodelling in bleomycin-induced pulmonary fibrosis. Senicapoc and tetrathiomolybdate treatments attenuated microvascular remodelling as well as pulmonary fibrosis. This is in contrast with the application of the FDA-approved drug pirfenidone, which only attenuated fibrosis, without any effects on the vasculature, in this model. It will be interesting to see whether intervention strategies derived from this thesis can be applied clinically to treat IPF.
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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.