Datta, G; (2014) Investigating the role of endothelial nitric oxide synthase in liver ischaemia reperfusion injury and direct ischaemic preconditioning using a transgenic double knockout model of endothelial nitric oxide synthase. Doctoral thesis , UCL (University College London).

Preview

PDF Datta_G_EDITED_3rd_PARTY_Copyright_REMOVED_2014_2014.pdf Download (2MB)

Abstract

Background: Liver ischaemia reperfusion injury (IRI) occurs after prolonged ischaemia followed by reperfusion in the clinical setting, such as liver resection surgery and liver transplantation and is associated with increased morbidity and mortality. Ischaemic preconditioning (IPC) is a therapeutic strategy to lessen liver IR injury. Liver IPC is a mechanical technique whereby a short period of occlusion of the blood supply to the liver confers protection against IR injury. Understanding the molecular mechanisms of IPC offers the possibility of developing techniques and pharmacological agents that will reduce IRI and improve clinical outcomes. Hypothesis, Aims and Objectives: The molecular mechanisms of liver IPC are not clearly established. Nitric oxide is an important mediator, but the role of the activation and expression of nitric oxide synthase (NOS) and its isoforms endothelial NOS (eNOS) and inducible NOS (iNOS) is unclear. Haem oxygenase-1 (HO-1) protects, but it is unclear if this depends on NOS. There appear to be two phasese of liver IRI and IPC: an early and late phase. There is evidence that NOS has roles in both phases, but that HO-1 has a role only in the late phase. The principal aim was to develop a transgenic eNOS knockout (eNOS-/-) model of liver IRI and IPC to specifically probe the in vivo physiological roles of eNOS and its interactions with HO-1 these processes. Methods: An in vivo mouse model of partial (70%) warm hepatic ischaemia reperfusion (IR) was used. Normal and transgenic double knockout for eNOS (eNOS-/-) mice were used. A partial warm liver IR model was used where ischaemia was applied to cephalic lobes only followed by reperfusion. Ischemic preconditioning (IPC) consisted of ischaemia applied directly to the cephalic lobes followed by reperfusion then IR to the cephalic lobes. Final reperfusion was either for 2 hours (normal and eNOS-/- mice) representing early phase IRI or for 24 hours (normal mice) representing late phase IRI after which the animals were terminated for tissue and blood samples. The endpoints measured were surface laser Doppler flow to assess liver 14 microcirculation during the experiment and blood and liver tissue samples at the end of the experiment for serum ALT, liver histological injury scores, Western blotting for eNOS, iNOS, phosphorylated eNOS (p-eNOS), HO-1 protein and RT-PCR for HO-1 mRNA. Results: 1. In this model of early phase partial hepatic IR consisting of 45 minutes ischaemia to the cephalic lobes and 2 hours reperfusion (index IR), there was IRI in normal mice across the three endpoints for IRI of serum ALT, histological injury and microcirculatory dysfunction. IPC consisting of 5 minutes ischaemia and 10 minutes reperfusion (IPC 5/10) preceding index IR reduced IRI across the three endpoints in normal mice. In eNOS-/- knockout mice, there was also IRI across the three endpoints, with greater hepatocellular and histological injury than normal mice, but no difference in the microcirculatory dysfunction compared to normal mice. In eNOS-/- knockouts IPC 5/10 did not reduce IRI across the three endpoints. This indicates that eNOS is a mediator of the protective effects of IPC in early phase liver IRI and baseline eNOS reduces the severity of IRI even without IPC. Based on the differences on the effects on the endpoints between all the experimental groups, it appears that IPC protection is mediated by eNOS in hepatocytes and sinusoidal endothelial cells (SECs) and baseline eNOS protection in liver IR without IPC is mediated by hepatocyte eNOS only. 2. It was demonstrated that in the early phase partial hepatic IR model that both IR and IPC increased eNOS protein expression and eNOS activation by phosphorylation with no additional effect of IPC. This indicates that the protective effect of eNOS with IR alone may at least be partly mediated by increased eNOS protein expression and activation by phosphorylation, but the benefits of IPC in reducing early phase IR injury are mediated by eNOS activation by other mechanisms. Expression of iNOS protein does not play a role in IR injury in our model. Haem oxygenase-1 (HO-1) protein was not expressed in our model, but HO-1 mRNA was expressed in both normal and eNOS-/- animals following liver IR and IPC, indicating that HO-1 expression is not dependent on eNOS. HO-1 may therefore still have a protective effect in the late phase of IR injury acting in a parallel pathway to eNOS. 15 3. In the model of late phase partial (70%) hepatic IR consisting of 45 minutes ischaemia, recovery from anaesthetic and 24 hours reperfusion (index IR) developed using normal mice only, there was liver IRI across two endpoints of serum ALT and histological injury (microcirculatory dysfunction was not studied in the late phase) with progression of histological injury from mainly sinusoidal congestion to hepatocyte necrosis. IPC consisting of 5 minutes ischaemia and 10 minutes reperfusion (IPC 5/10) preceding index IR reduced IRI across the endpoints. HO-1 protein was detected with late phase IR and IPC, indicating timecourse of early phase HO-1 mRNA expression followed by late phase HO-1 protein expression. This is consistent with the possibility of HO-1 potentially having a protective role in late phase liver IR and IPC. Conclusions We have developed a model of early and late phase liver IRI and IPC, which has demonstrated that eNOS is a protective mediator in IRI and IPC in the early phase, HO-1 is activated independently of eNOS and may have a role in late phase IRI and IPC. This model should prove useful in investigating the roles of eNOS and HO-1 in liver IPC and IR injury. This would ultimately be used to identify pathways for development of pharmacological agents that would effectively reduce liver IR injury in the clinical setting and improve patient outcomes related to this.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as CSVXMLJSON

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item