PRUEBAS DE INTEGRACIÓN

The results of the studies presented in this thesis highlight the relevance of immunohistological analysis of ICAM-1 expression in experimental animal models and in normal human livers as a consequence of ischaemia / reperfusion injury. The characteristic expression patterns of ICAM-1 due to ischaemia, ischaemia/reperfusion injury and post-transplantation were correlated with histological and functional parameters, which were used in this thesis to assess the reperfusion injury. Also the possibility was tested that ICAM-1 expression might act as a predictive marker for acute cellular rejection.

In Chapter 3, it was shown that up to 30 minutes of warm ischaemia did not induce significant up-regulation of ICAM-1 expression, however warm ischaemia of 45 minutes or more increased ICAM-1 expression on sinusoidal endothelial cells. It was reported that extent of warm ischaemia to 60 minutes or more affect the survival outcome in experimental liver transplantation (Ikeda eta!., 1992). Ischaemia/reperfusion resulted in a highly significant up- regulation of ICAM-1 expression on both sinusoidal endothelial cells and hepatocytes. Ischaemia/reperfusion had a more significant effect on ICAM-1 expression than the effect of ischaemia only. The systemic effects of reperfusion injury were evident in Chapter 3, where significant up-regulation in ICAM-1 expression in the non-ischaemia lobe was observed. It was also shown that up to Bhr cold ischaemia had no influence on ICAM-1 expression on sinusoidal endothelial cells, whilst extended cold ischaemia to 16hr or more induced a significant increase in ICAM-1 expression. In vitro liver reperfusion resulted in a significant up-regulation of ICAM-1 expression which

was more prominent when cold ischaemia times extended to 16hr or more. The beneficial effect of the free oxygen radical scavenger, reduced glutathione added to the liver perfusate was investigated in an attempt to modulate the effect of ischaemia/reperfusion injury and possibly ICAM-1 expression. It was demonstrated that there was no change in ICAM-1 expression in livers treated with GSH with limited cold storage time up to 8hr when compared with controls. It was observed that ICAM-1 expression was more pronounced in the untreated group when compared with the expression in the treated group following longer cold storage periods.

Warm ischaemia mainly affected the parenchymal cells (hepatocytes) and cold storage mainly affected non-parenchymal cells sinusoidal endothelial cells. In Chapter 4 it was shown that the morphological changes of the sinusoidal endothelial cells due to cold ischaemia were directly correlated with ICAM-1 expression. However, there was no correlation between morphological changes due to warm ischaemia and ICAM-1 expression. These observations might suggest that the injury due to cold ischaemia is not merely delayed warm ischaemic injury .

Reperfusion injury resulted in morphological changes in both sinusoidal endothelial cells and hepatocytes which were more severe than those from cold or warm ischaemia alone. This injury was directly correlated with ICAM- 1 expression. Treatment with GSH resulted in limited reperfusion injury in livers which had cold ischaemia up to 8hr. However, with longer ischaemia times GSH has no effect on such injury.

In Chapter 5 reperfusion injury was assessed by ALT and A S T levels and was noticeable after 30 minutes of warm ischaemia. The severity of the dam age was correlated with the length of the ischaemic period, as there was a significant peak in ALT and A S T levels after 60 minutes of warm ischaemia. Ischaemia/reperfusion resulted in significant increase in ALT and AST levels which were most marked with longer ischaemic time. The increased ALT and AST levels after reperfusion were more significant when compared with the effects of ischaemia only. It was also shown that there was a significant correlation between ICAM-1 expression and increased levels of ALT and AST after ischaemia and ischaemia/reperfusion. However, it was

noticed that reperfusion injury resulted after 30 minutes of warm ischaemia and ICAM-1 expression up-regulated after 45 minutes of warm ischaemia. This might explain why reperfusion injury proceeded the up-regulation of ICAM-1.

In Chapter 6 there was an observed reduction in hepatic perfusion during the warm ischaemia periods which was associated with increased ICAM-1 expression on the sinusoidal endothelial cells in liver biopsies that were taken at the end of the ischaemia times from the same ischaemic lobes. Ischaemia/reperfusion was also resulted in a reduction in flow of the hepatic microcirculation. The reduction in flow increased as the warm ischaemia time was extended. The reductions in hepatic microvascular perfusion during ischaemia and ischaemia/reperfusion correlated with the increase of ICAM-1 expression at the end of the ischaemia and ischaemia/reperfusion times. The correlation between the reductions in hepatic flow and increases in ICAM-1 expression might be explained by the effect of Ischaemia/reperfusion on Kupffer cells. Activated Kupffer cells release inflammatory cytokines which activate the sinusoidal endothelial cells and hepatocytes to express ICAM-1 on their membrane surfaces and also to release leukocyte-activating factors such as interleukin-1 or platelet-activating factor. These induce LFA-1 on the surface of neutrophils, resulting in the adhesion of ICAM-1 and LFA-1. Thrombi containing neutrophils may then be formed in hepatic sinusoids, causing tissue microcirculatory failure at the inflammatory sites (Nakano et a i, 1995).

In Chapter 7, in the field of human OLT, it was shown that extended cold ischaemia time beyond 12hr increased post-operative dam age in hepatocytes as assessed by ALT, AST, bilirubin levels and prothrombin time. However, there was no correlation between cold ischaemia times and the incidence of early and late graft rejection. It was also found that warm ischaemia time had no effect on the degree and the incidence of hepatocyte injury. However, there was a significant correlation between the length of the warm ischaemia time and the incidence of acute cellular rejection during the first week post-transplantation.

allograft acute cellular rejection. The addition of GSH to flush-out solution did not improve the ischaemia/reperfusion injury or decrease the incidence of acute cellular rejection in the treated group.

In Chapter 8, harvesting procedures did not stimulate the immune system on the basis of adhesion molecule staining in the donor allografts, as there were no differences in ICAM-1 expression in the harvested allografts (after in situ flush) when compared with expression in baseline biopsies. There was also no significant effect of cold ischaemia time on the intensity of ICAM-1 staining. During OLT, ICAM-1 induction on sinusoidal endothelial cells and hepatocytes was recorded at a very early phase of immune activation (90 minutes post­ reperfusion). However, no correlation was observed between up-regulated ICAM-1 expression in post-reperfusion biopsies and the incidence of ischaemia/reperfusion injury. Freshly prepared GSH was added to the allograft flush out solution just before starting reperfusion. ICAM-1 expression was significantly noticeable in the untreated group when compared with the GSH treated group. It was also shown that post-reperfusion ICAM-1 expression correlated with early and late acute cellular rejection.

Examination of the post-reperfusion allograft liver biopsies by light microscop after immunohistochemical staining could be a useful screening test to help in early prediction of acute cellular rejection.

In this thesis some functional parameters, may be more affected than others. However, collectively the data in these studies indicate that in the field of liver transplantation, all liver allografts experience periods of warm and cold ischaemi, injury followed by reperfusion injury. It may be that different protective strategies for periods of either warm or cold ischaemia will be required to optimise results of liver transplantation.

In summary, it can be stated that clinical liver preservation and transplantation is accompanied by stimulation of adhesion molecules as part of the inflammatory response. This was not only a result of the allograft response, because, in the experimental liver perfusion studies, ICAM-1 expression was up-regulated after cold storage even when reperfusion was carried out using a bloodless aqueous perfusate (Krebs solution). However, This sensitisation

of the immune system by ischaemia/reperfusion injury was associated with a demonstrable increase in the severity of the allograft response, leading to greater incidences of early and late acute rejection episodes. The results presented in this thesis therefore are additional support for the hypothesis that inflammation, arising from either of these two routes, combine to have a negative impact on the outcome of transplantation (Land eta!., 1999).

Whilst oxygen free radicals are generally implicated in the inflammatory process, addition of the scavenging agent reduced glutathione had only marginal effects on ICAM-1 expression. This suggests that the agent was either unable to deal with all the possible sources of oxygen free radicals (as a water soluble agent it may not be readily accessible to sources of oxygen free radicals generation within hydrophobic sites in the cells such as membrane bilayers), or was not present at the right time (it may have been needed to be present in the reperfusing blood, rather than only in the washout solution). On the other hand, the fact that GSH had any affect at all gives some hope that in the future a better therapy can be found.

Thus for the future, several areas are highlighted for further research on adhesion molecule expression. Firstly, further work should be aimed at investigating the early events which play a role. Activation of nuclear transcription factors such as nuclear factor-KappaB (NF-kB) are thought to be central to inflammatory responses (Rahman & MacNee., 2000), and it might be more useful to target these early molecular changes, rather than attempting to influence measurable ICAM-1 protein by immunohistochemistry, which may be already too late to stop interactions with circulating leukocytes. Another approach would be to use a combination therapy of drugs which could act both in the intra- and extra-cellular environments. Reduced glutathione, whilst being a clinically acceptable agent, does not penetrate cell compartments easily. Finally, improved preservation methods should be investigated to overcome the ischaemic cascade which leads to ischaemia/reperfusion injury. It is being consistently demonstrated that perfusion technologies provide enhanced aerobic renal preservation (Burdick e ta l., 1997). However, this has yet to be transferred to other organs such as the liver. Whatever route is taken, it is clear that a full understanding of

inflammatory responses to ischaemia/reperfusion injury will yield benefits for patient outcomes in the future.

List of Publications

Expression of ICAM-1 during Orthotopic liver transplantation.

El-Wahsh M, Fuller BJ, Sreekumar NS, Burroughs A, Dhillon P, Rolles K, Davidson BR. Biochemical Society Transaction] 25: 1997.

Effect of reperfusion injury on Human Allograft ICAM-1 Expression and Its Correlation with Histological Evidence of Reperfusion Changes.

El-Wahsh M, Fuller BJ, Sreekumar NS, Burroughs A, Dhillon P, Rolles K, Davidson BR. Transpiant.Proc]29-A997.

Correlation of ICAM-1 expression with post-operative acute cellular rejection in Human Liver Allograft.

El-Wahsh M, Fuller B, Sreekumar NS, Dhillon P, Burroughs A, Rolles K, Davidson BR. Biochemical Society Transaction;25:^997.

Effect of free radical scavenger (reduced glutathione) on PECAM expression in Liver Transplantation.

El-Wahsh M, Fuller B, Sreekumar NS, Dhillon P, Burroughs A, Davidson, BR, Rolles K. Biochemical Society Transaction',25-A997.

Adhesion molecule activation and hepatocyte damage following orthotopic liver transplantation.

El-Wahsh M, Fuller B, Sreekumar NS, Dhillon P, Burroughs A, Rolles K, Davidson BR. Biochemical Society Transactions,26^,^ 998.

Does Glutathione flush reduce acute cellular rejection in liver allografts?

M El-Wahsh, B Fuller, N Sreekumar, P Dhillon, A Burroughs, B Davidson, K Rolles, Biochemical Society Transactions ,26; 1998.

Effect of reduced glutathione on PECAM expression in human liver allografts. M El-Wahsh, B Fuller, N Sreekumar, P Dhillon, A Burroughs, B Davidson, K Rolles. HPB Journal. 1,1997; 107 (Abstract)

Correlation of ICAM-1 expression with histological assessment of reperfusion injury. M El-Wahsh, B Fuller, N Sreekumar, P Dhillon, A Burroughs, K Rolles, B Davidson. HPB Journal, 1, 1997; 122 (Abstract)

Effect of reduced Glutathione on Intercellular Adhesion Molecule-1 expression in Liver Allografts.

M El-Wahsh, B Fuller, N Sreekumar, P Dhillon, A Burroughs, B Davidson, K Rolles. Gut, (Supl 3), 41, 1997; A240 (Abstract)

Is preservation injury predictive of early acute cellular rejection in liver allografts ? M El-Wahsh, B Fuller, N Sreekumar, P Dhillon, A Burroughs, K Rolles, B Davidson. Gut, (Supl 3), 41, 1997; A240 (abstract)

Soluble ICAM-1 is released from the liver in end stage cirrhosis. M El-Wahsh, V Shah, B Fuller, A Burroughs, K Rolles, BR Davidson. HepatoGastroenterology {Sup\ II), 4 5 ,1 9 9 8 , OX (Abstract)

Free Oxygen radical scavenger does not improve graft function following Orthotopic Liver Transplantation.

M El-Wahsh, B Fuller, A Burroughs, BR Davidson, K Rolles. HepatoGastroenterology {Sup\ II), 45, 1998, GCCLVI (Abstract)

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