Agrawal, A.; (2010) Optimizing pancreas preservation for islet transplantation: mechanisms and bioenergetics of the two-layer method. Doctoral thesis , UCL (University College London).

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Abstract

Background: The recent unprecedented interest in islet allotransplantation has been tempered by the observation that sustained freedom from exogenous insulin is rarely achieved and the rate of insulin independence drops to 10% at 5 years follow-up. One critical determinant of successful islet allografting is preservation injury to the pancreas. The two-layer method (TLM) of pancreas preservation was developed to mitigate the deleterious effect of cold ischaemia, but the mechanism is unclear and its clinical efficacy is controversial. Hypothesis, Aims and Objectives: There is no clinical or experimental evidence for benefit of the two-layer method as currently employed in pancreatic islet transplantation. A potentially beneficial effect on pancreas preservation by improvement in graft ATP production is possible by modification of the two-layer method. The principal aim was to develop optimal protocols for pancreas preservation in islet transplantation by clarifying the mechanism of TLM. A second objective was to develop a dynamic model for the study of mitochondrial function during organ preservation. Methods: 1. Perfluorocarbon (PFC) content of porcine pancreases preserved in TLM and in University of Wisconsin (UW) solution for 24 hours was compared. Pancreatic samples were analysed using Varian INOVA 9.4T MR scannerspectrometer. External PFC standard was introduced for quantification. Four consecutive transverse images of 4mm thickness were obtained using a spin-echo sequence. 19F MRS was performed with the same parameters except with more averages. MR data was confirmed by headspace chromatography. 2. Real-time changes in pancreas bioenergetics were studied with 31P MRS for rat pancreases preserved at 4°C - 6°C in five different groups: chilled Marshall‟s, static TLM, continuous TLM with oxygen perfused at 0.5L/h, and static or continuous TLM both the latter following 30 min warm ischaemia. 31P spectra were analyzed for phospho-mono-esters, inorganic phosphate (Pi) and α-, β- and γ-nucleotide triphosphate. Results: 1. PFC standard was readily detected in 19F MR images. There was no signal from porcine pancreas in 19F MR images following either UW or TLM storage. 19F MR spectra typical of PFC were not obtained from either UW-or TLM-preserved pancreas with non-localized 19F MRS. Mean concentration of PFC in TLM pancreas measured by head space chromatography was not significantly different from background concentration in UW pancreas. 2. Intergroup rates of change of [γ-ATP]/[Pi] and [β-ATP]/[Pi] throughout preservation period were significantly different. For continuous TLM there was an increase relative to baseline but decrease for both static TLM and Marshall‟s with respect to continuous TLM. Rate of decrease was similar for the Marshall's and static TLM groups. [γ-ATP]/[Pi] and [β-ATP]/[Pi] increased with WI continuous TLM but decreased for WI static TLM. Conclusions: There is no evidence of penetration of perfluorocarbon into pancreas tissues investigated either by MR or chromatography in organs preserved at hypothermia. 31P-MRS is an effective tool for non-invasive assessment of pancreas bioenergetics. Continuous TLM preserves cellular bioenergetics and is superior to current non-PFC based solutions for pancreas preservation.

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