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The role of nitric oxide in hindlimb remote ischaemic preconditioning of the liver

Abu-Amara, M.; (2011) The role of nitric oxide in hindlimb remote ischaemic preconditioning of the liver. Doctoral thesis, UCL (University College London). Green open access

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Abstract

Ischaemia reperfusion (IR) injury is a major contributor to morbidity and mortality following liver resection and transplantation. Hindlimb remote ischaemic preconditioning (RIPC) reduces liver IR injury but the mechanisms underlying this protection are unknown. This thesis evaluated the effects of RIPC on the early phase of liver IR injury in a new mouse model. This model was used to assess the role of the nitric oxide (NO) pathway in mediating the protective effects of RIPC on liver IR injury. In the initial experiments wild type C57BL/6 mice were used to establish a new model of hindlimb RIPC that protects against liver IR injury. The new experimental model hindlimb protocol consisted of 6 cycles of 4 minutes of femoral vessel clamping followed by 4 minutes of reperfusion. The IR protocol consisted of a well-described 40 minutes of lobar liver (70%) ischaemia followed by 2 hours reperfusion. This hindlimb RIPC model resulted in significant reductions in liver IR injury, as evaluated by plasma liver transaminases levels, histopathological scores, and ultrastructural assessment of cellular damage in the liver. In addition hindlimb RIPC preserved the hepatic microcirculatory blood flow (MBF) in livers subjected to IR. In subsequent experiments administration of the selective NO scavenger C-PTIO to wild type mice abrogated the protective effects of limb RIPC on liver IR injury. In addition the RIPC-induced preservation of hepatic MBF was attenuated by C-PTIO administration. In contrast to the protective effects of limb RIPC on liver IR injury in wild type mice; mice lacking the constitutively expressed NO synthase (eNOS-/-) enzyme were not protected against liver IR injury by hindlimb RIPC, and MBF measurements in these mice showed no benefit of RIPC on the microcirculation. In wild type mice hepatic and limb eNOS protein expression was similar among preconditioned and non-preconditioned animals. In comparison expression of the inducible NOS (iNOS) isoform was only seen in preconditioned animals. In order to elicit the pathway through which RIPC-derived NO protects against liver IR injury, plasma nitrite and nitrate (NOx) levels were quantified in wild type animals and shown to be significantly elevated in preconditioned compared to non-preconditioned animals. However, intravenous administration of exogenous nitrite to eNOS-/- mice undergoing liver IR injury failed to mimic the protective effects of RIPC-derived endogenous NOx in wild types. Administration of the soluble guanylyl cyclase (sGC) inhibitor ODQ showed a trend indicating reversal of the protective effects of RIPC on liver IR injury but this did not reach statistical significance. ODQ significantly annulled the protective effects of limb RIPC on hepatic MBF during liver reperfusion. Measurement of hepatic cyclic GMP levels in wild type animals showed a significant increase in animals subjected to limb RIPC only compared to sham. However there was no difference in the cGMP levels in the RIPC + IR compared to the IR group. In conclusion this thesis has described a new mouse model of limb RIPC that protects against liver IR injury. It was also shown that NO and eNOS are essential in mediating the protective effects of limb RIPC on liver IR injury. Endogenous NOx metabolites of NO play a crucial role in RIPC induced protection. The hepatic sGCcGMP pathway is at least partially involved in RIPC-induced liver protection.

Type:Thesis (Doctoral)
Title:The role of nitric oxide in hindlimb remote ischaemic preconditioning of the liver
Open access status:An open access version is available from UCL Discovery
Language:English
UCL classification:UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Surgery and Interventional Science (Division of)

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