An Ex Vivo Normothermic Porcine Pancreas Physiological Model: Implications for Pancreas and Islet Transplantation
2017-09-25T15:07:55Z (GMT) by
Introduction: The aim of the work performed for this thesis was to establish an ex vivo normothermic porcine pancreas perfusion model which is physiological and has the potential to facilitate detailed study of both its exocrine and endocrine function. The ex vivo porcine pancreas model was used to investigate the effects of machine perfusion on parameters that may be important in graft preservation and the success of whole organ pancreas and islet transplantation. A porcine islet isolation and digestion protocol was also established to enable the assessment and quantification of islet yield and viability. Methodology: During the preliminary phase of the thesis eight porcine pancreata were recovered and perfused in order to establish a reproducible experimental protocol for an ex vivo physiological pancreas model. Subsequently nine porcine pancreata were successfully recovered and normothermically perfused with autologous blood at a mean blood pressure of 50 mmHg (normotensive). Graft viability was then compared against a further four ex vivo porcine pancreata normothermically perfused at 20 mmHg (low) pressure. Serological and haematological parameters measured were: arterial and venous oxygen gas differential, routine biochemistry, glucose concentration and graft insulin responses to glucose stimulation. Exocrine function was assessed by measuring the rate of production of pancreatic juice (volume/time) and the level of serum amylase. Immunohistochemistry for cellular viability was assessed by haematoxylin and eosin staining, M30 cytoDEATH, anti-Caspase 3 antibody, and anti-ATP synthetase complex V antibody. Results: All normotensive pancreata were perfused for a median of 3 hours (range 2–4 hours) with a mean perfusion pressure of 50 mmHg and graft flow rate of 141 mL.min-1 (95% confidence intervals 122.4 to 160 mL.min-1). In comparison, all of the ‘low’ pressure models were successfully perfused for a minimum of 4 hours, with mean perfusion pressure of 20 mmHg and graft flow rate of 40 mL.min-1 (95% confidence interval 31 to 48 mL.min-1), p <0.05. All pancreata in both the normotensive and low pressure groups demonstrated cellular viability with evidence of oxygen consumption and preserved endocrine and exocrine function. Following statistical analysis, the ‘low’ pressure perfused porcine pancreata compared favourably in important biochemical and immunohistochemistry cellular profiles, suggesting the potential for an improved method of graft preservation with improved viability. Conclusion: The physiological behaviour of this ex vivo perfused pancreas model allows the changes that occur in recovered pancreata and the potential effect they have on islet isolation, yield and whole organ viability to be studied in detail. The model also avoids use of live animals, is reproducible and mimics a “donation after circulatory death” pancreas transplant scenario. The work in this thesis demonstrated the successful application of the model to investigate the effect of machine perfusion on variables that may influence and potentially optimise both a whole organ pancreas and islets prior to transplantation.