Cellular and Molecular Mechanisms of Pancreatitis in Atrophy and Regeneration
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Cellular and molecular mechanisms of pancreatitis in atrophy and regeneration Lynne Estelle Reid BSc, MSc (Qual) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Faculty of Medicine i ABSTRACT Pancreatitis is a debilitating disease with complex etiology, prognosis and pathology, limited treatment options and a burden on health resources. The main lesions of acute pancreatitis are injury to acinar cells and inflammation of the pancreas. Mild interstitial pancreatitis (or oedematous pancreatitis) is characterized by acute inflammation with oedema in the stroma and acinar tissue. Necrosis of acinar tissue, stroma or peri-pancreatic fat is absent and complete recovery, or regeneration of the gland is usual. Whilst there is universal agreement on many of the cellular changes that accompany acute pancreatitis, there is no clear distinction between the factors that determine whether regeneration can occur. The mechanism of regeneration and the cellular processes that influence regeneration form the primary basis of this study. Experimental models of pancreatitis have been produced in rats, mice, dogs, cats and swine by various methods. The time course and final tissue regeneration, if it occurs, vary considerably among different models, with many having the limitation that pancreatic injury is extended, so that degenerative processes are superimposed on regenerative activity. Three in vivo models of pancreatitis in rats were chosen for this project, to study pancreatitis and pancreatic regeneration that occurs in the recovery phase. These models evolve slowly, allowing detailed morphological study. In general, they have similar early events but vary considerably in regenerative outcome. (1) The infusion of caerulein is a well-established model for inducing experimental pancreatitis in rats and pancreatitis is followed by a great reparative capacity. Caerulein is a decapeptide with biological activity on gastrointestinal smooth muscle contraction and pancreatic and gastric secretion. (2) In 1-cyano-2-hydroxy-3-butene (CHB)-induced pancreatitis, acinar cell regeneration is limited to small islands of acini which fail to endure. CHB is known to induce cell death in pancreatic acini. (3) The model of partial pancreatic duct ligation leads to atrophy of the gland with little or no regeneration ensuing. Although some characteristics are known about these models of pancreatitis, there is a general lack of information on the association between pancreatic atrophy and inflammation and investigating this association was one of my overall aims. In addition, as well as investigating the three different models of pancreatitis in rats, I aimed to compare the same initiating event in other animals. Because of the selective nature of CHB pancreatotoxicity, and the ease of producing the model in rats, I sought to investigate this method in a mouse model. In addition, I sought to establish and characterise a canine CHB model. ii The pancreatic atrophy induced in the chosen models typically involved apoptosis of acinar cells and its removal by macrophages. In some situations, if acinar redifferentiation did not occur, development of fibrosis and scarring was a key to pancreatic repair. This atrophy took place in a coherent fashion with normal architectural relationships being maintained. Loss of the potentially dangerous enzyme- secreting cells by apoptosis led to regression of the underlying duct structure such that regeneration might occur should the circumstances change. This thesis has had a prolonged history because of part-time enrolment, through departure from the University of Queensland of my original Principal Advisor Dr Neal Walker, transfer of my Associate Advisor Dr Lyndell Kelly to Dunedin, New Zealand and necessary changes in my Advisory Team, an attempt to complete some of the research in New Zealand and the unfortunate death of the scientist I was working with, as well as my own personal and family issues. I am hoping to complete the project with my current re-enrolment. The hypothesis tested was that different models of pancreatitis can be used to describe the processes of atrophy and regeneration in the pancreas. The specific aims of this study were: 1. To investigate rat models of pancreatitis using caerulein, CHB and duct ligation; 2. To validate a pancreatitis model with CHB in the mouse; 3. To establish a canine model of pancreatitis with CHB; 4. To compare and contrast the models with respect to: Stellate cells and the production of fibrosis The role of mast cells and macrophages The role of survivin in pancreatic regeneration Summary of outcomes of this study Study of the three rat models of pancreatitis facilitated a comparison of markers and mechanisms within the lesion. All of the pathological processes studied, including fibrosis, stellate cells, mast cells and macrophages had specific roles in the different models that correlated with the initiation of pancreatitis, the deposition and degradation of fibrosis, and the ability of the pancreas to regenerate. Resorption of fibrosis may depend on the type of collagen deposited. Survivin appears to be maintaining conditions for the survival of cell populations. The models of CHB in mouse and dog were not suitable for the study of pancreatitis as the liver showed more severe damage at low doses than the pancreas iii DECLARATION BY AUTHOR This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. iv ACKNOWLEDGEMENTS I acknowledge and thank those who have given me support throughout this long journey. Prof Neal Walker, now Envoi Pathology, Brisbane, introduced me to pancreatitis as supervisor for my Masters Qualifying thesis and was first supervisor for the PhD thesis. Prof Grant Ramm (QIMR-Berghofer Research Institute) supervised a comprehensive but ultimately unsuccessful and aborted search for the elusive pancreatic stem cell. Dr Leila Cuttle (then, UQ School of Medicine) taught me how to use the ImagePro Plus® image analysis software. Dr Richard Malik (Centre for Veterinary Education, University of Sydney) and Dr Melanie Latter (veterinary pathologist, formerly UQ School of Veterinary Science) provided veterinary proficiency. Dr Anne Marie McNicol (now retired, Reader in Pathology, Glasgow University) perused some of the earlier manuscript. Work colleagues from the past, Clay Winterford, Estelle Schoch and from the more recent past, the brilliant staff too numerous to mention, of the Lakhani Breast Cancer Research Group, are sincerely thanked. Debates and discussions about research and protocols, sharing of knowledge and general moral support, were invaluable. Dr Lyndell Kelly (School of Medicine, University of Otago, New Zealand) has been a friend and colleague extraordinaire and a PhD supervisor: The Three Musketeers, Lyndell, Lynne and Pancreas have planned projects, finished some, made the acquaintance of lots of laboratory animals and enjoyed mutual scientific discovery. I could not have done this without you. A/Prof Glenda Gobe (School of Biomedical Sciences, UQ Faculty of Medicine), I am forever grateful for your expertise, patience and good humour in helping me achieve the finished product. v PUBLICATIONS Peer-reviewed publications during candidature Reid L and Walker N. Acinar cell apoptosis and the origin of tubular complexes in caerulein- induced pancreatitis. Int. J. Exp. Pathol. 1999, 80, 205-215. Kelly LE, Reid L, Walker NI. Massive acinar cell apoptosis with secondary necrosis, origin of ducts in atrophic lobules and failure to regenerate in cyanohydroxybutene pancreatopathy in rats. Int. J. Exp. Pathol., 1999, 80, 217-226. Peer-reviewed published abstracts during candidature Kelly LE, Goodall AR, Reid L, Walker NI. The effect of the pancreatotoxin cyanohydroxybutene on pancreatic ductal and acinar carcinoma cell lines. In Proceedings of the 5th International Symposium on predictive Oncology and Therapy, Cancer Detection