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Background – Feeding horses round bale hay (RBH) has been associated with airway inflammation. The purpose of this study was to determine if horses fed RBH for a 6-week period demonstrated more evidence of airway inflammation than horses fed square bale hay (SBH) of comparable quality. Hypothesis - The respiratory health of horses fed RBH will not differ from horses fed SBH of comparable quality. Animals – Two feeding groups of 15 healthy horses (mixed ages, breeds) from the University riding program. Methods – This was a prospective study performed during fall of 2009. At the beginning and end of a 6- week feeding trial, horses were examined (physical, upper airway endoscopic) and samples (tracheal aspirate (TA), bronchoalveolar lavage (BAL)) collected for cytology and/or bacterial/fungal culture. Hay was analyzed for nutritional value and bacterial/fungal content. Results – Horses fed RBH demonstrated an increase in pharyngeal lymphoid hyperplasia (p=0.0143) and percentage neutrophils (p=0.0078) in the TA samples post-feeding as compared to pre-feeding values. Nutritional analysis of hay and measurements of bacterial/fungal load did not differ over time and/or between hay types. Conclusions and clinical importance – The identification of airway inflammation in the horses fed RBH indicates that factors associated with the manner in which the hay is fed and consumed contribute to the development of subclinical airway inflammation. RBH affords horses continuous daily exposure to hay and as horses bury their muzzles in the bale, exposure to particulate matter is likely increased. These factors may partially explain the response in horses fed RBH. Further studies are required to confirm these predictions. / Master of Science

Verschiedene Ursachen erfordern rekonstruktive Maßnahmen an der Trachea zur Erhaltung eines suffizienten Luftweges. Häufig treten im Rahmen dieser Eingriffe Infektionen und Schädigungen auf, die die Bildung von Granulationsgewebe nach sich ziehen und zu Stenosen führen können. Der Einsatz von epithelialisierten autogenen oder auch allogenen Transplantaten, die mit der Methode des Tissue Engineering hergestellt werden, bietet einen neuen Lösungsansatz, um Stenosen zu vermeiden. Diese Arbeit beschäftigt sich mit der Isolierung, Kultivierung und Charakterisierung von humanem respiratorischen Epithelzellen (hREC), sowie deren Einsatz in Co-Kulturen mit humanen Chondrozyten als einen ersten Schritt zur Transplantatherstellung. Die hREC wurden sowohl in nativem Gewebe als auch in Monolayerkultur und in verschiedenen Differenzierungkulturen histologisch und immunhistochemisch analysiert. Zusätzlich wurde die Ziliogenense mit der Elektronenmikroskop untersucht. Eine weitere Charakterisierung erfolgte durch die Genexpressionsanalyse einiger Cytokeratine auf RNA-Ebene mit der semiquantitativen real-time RT-PCR. Mittels Durchflusszytometrie konnten Basalzellen, die auch als Vorläuferzellen des humanen respiratorischen Epithels gelten, mit den Antikörpern CD49f und CD104 detektiert und analysiert und unter Verwendung der fluoreszenzaktivierten Zellsortierung (FACS) separiert werden. Es zeigte sich, dass die hREC in den Proliferationskulturen dedifferenzierten und durch spezielle Basalzellmarker angefärbt wurden. Die Differenzierungskulturen und ALI-Kulturen gaben erste Hinweise auf die Differenzierung der Zellen. In den Co-Kulturen konnte unter dem Einfluß eines Air-Liquid-Inteface ebenfalls eine Re-differenzierung der Zellen beobachtet werden. Die Ergebnisse zeigen, dass es möglich ist, eine Epithelialisierung von kollagenbeschichteten Biomaterialien oder auch autologem Knorpel zu erreichen, um diese Konstrukte für das Trachea Tissue Engineering einzusetzen. / The replacement of extensive tracheal defects resulting from intensive care medicine, trauma, or large resections is still challenged by the re-epithelialization of an autologous or alloplastic trachea replacement. Therefore, this thesis was performed to investigate the potential of culture expanded human respiratory epithelial cells (hREC) to regenerate a functional epithelium for trachea tissue engineering.hREC from nasal turbinates were freshly isolated, expanded and subsequently cultured in high-density multilayers to allow epithelial differentiation. Composition of epithelial cells in native respiratory epithelial tissue and culture expanded hREC were analyzed by histological staining and by immunohistochemical staining with the specific antibodies. Differentiation of culture expanded hREC was further characterized by gene expression analysis of a cytokeratin pattern using semi-quantitative real-time RT-PCR technique. Furthermore, basal cells known as progenitors of the respiratory epithelium were seperated by Fluorescense Activated Cell Sorting with the basal cell specific antibodies CD49f and CD104. Co-cultures of hREC and human chondrocytes (hCHO) or human cartilage respectively were compared to Air-Liquid-Interface cultures containing hREC and hCHO.Histological and immunohistochemical staining and Scanning Electron Microscopy pictures of hREC in differentiation cultures demonstrated basal cells covering the collagenous matrix. These cells formed a cellular multilayer, which is composed of a basal layer of undifferentiated basal cells and an upper layer of cells differentiating along the squamous metaplasia and ciliated cell lineage. Lineage development of cultured hREC was further documented by the induction of specific cytokeratins. Our results suggest that culture expanded hREC have the potential to colonize collagen coated biomaterials as well as autologous cartilage grafts and to regenerate epithelial cell types for trachea tissue engineering.

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INTRODUCTION. Congenital diaphragmatic hernia (CDH) combines a congenital malformation of the diaphragm with lung hypoplasia, leading to severe respiratory distress and intractable pulmonary hypertension of the newborn. Despite advances in prenatal diagnosis and neonatal intensive care, CDH is associated with high mortality and devastating morbidities. In the absence of curative treatment, numerous prenatal therapies have been used experimentally with varying success. So far, only fetal tracheal occlusion has been tested in clinical trials, but the consequences for the human lung are poorly known. AIMS. To further characterize the rabbit model of CDH, which was subsequently used to assess the effects of prenatal therapies on airway and pulmonary vascular development, including tracheal occlusion, and two novel approaches, perfluorooctylbromide and an activator of soluble guanylate cyclase (BAY 41–2272), which were given through tracheal instillation.METHODS. After a diaphragmatic incision during the pseudoglandular stage, fetal rabbits were randomized against placebo/sham operation during the saccular stage for tracheal occlusion, perfluorocarbon or BAY 41–2272. At term operated fetuses and controls were subject to evaluation of lung mechanics and/or hemodynamics as well as postmortem lung analyses. Human fetal and neonatal lung tissue, including controls and CDH with tracheal occlusion or expectant management, was analyzed histologically and biochemically.RESULTS. The rabbit model of CDH was characterized by reduced lung volumes and impaired compliance, disorders of elastin deposition within alveolar walls, and downregulation of elastogenesis-related genes. Moreover, this model reproduced features of pulmonary hypertension, including high right ventricular pressure and level of N-terminal-pro-B type natriuretic peptide, remodeling of pulmonary arterioles, decreased alveolar capillary density, and downregulation of vasodilation-related genes. In the rabbit model, lung distension caused by tracheal occlusion improved alveolar formation and elastogenesis, yet without correction of lung mechanical parameters. Tracheal occlusion increased also the expression of other extracellular matrix components, which reflected myofibroblast activity, and reduced the transcription of surfactant-associated proteins. Human neonatal lungs exposed to fetal tracheal occlusion displayed alveolar deposits of collagen and myofibroblasts. In human CDH as well as in the rabbit model of CDH, tracheal occlusion enhanced the pulmonary expression of transforming growth factor-β (TGFβ) and Rho kinase−associated proteins to the detriment of activation of SMAD2/3, which is normally detected in human lungs with advancing gestation. As an alternative to tracheal occlusion, pulmonary distension by perfluorocarbon in the fetal rabbit model of CDH improved lung mechanics and alveolar elastogenesis without transcriptional changes in extracellular matrix, surfactant protein genes or TGFβ. Finally, intratracheal instillation of BAY 41–2272 in the rabbit fetuses with CDH improved hemodynamics, reduced medial hypertrophy of pulmonary arterioles, and increased capillary bed formation by stimulating endothelial cell proliferation.CONCLUSIONS. In the fetal rabbit model of CDH, poor lung function after tracheal occlusion is compatible with activation of TGFβ and imbalance in extracellular matrix and epithelial homeostasis. In human CDH newborns treated by fetal tracheal occlusion, changes in the pulmonary interstitium and impaired TGFβ signaling raise the question of disturbances of postnatal lung development induced by tracheal occlusion. As potential alternatives to tracheal occlusion, prenatal perfluorocarbon improves lung hypoplasia, whereas prenatal BAY 41–2272 attenuates pulmonary hypertension. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

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