Overall conclusions arising from the thesis and future plans

The results of the study have been discussed in detail in each chapter detailing the experiments (Chapters 4 to 7). This section addresses an appraisal of the overall conclusions and the neccessity and design of future studies are discussed.

The major findings of this study in the rat model of lober liver ERJ were:

increase in nitric oxide production.

2. The increase in nitric oxide was observed immediately after preconditioning period and continued through the subsequent ischaemia and reperftision periods. 3. With ischaemic preconditioning, nitric oxide synthase was expressed by

hepatocytes and vascular endothelium; eNOS was upregulated whereas iNOS expression remained absent suggesting that eNOS derived nitric oxide was associated with the ischaemic preconditioning effect.

4. The protective effect of ischaemic preconditioning was applicable to the fatty liver.

The above conclusions support the hypothesis presented in this study and strongly suggest a role for eNOS derived NO in hepatoprotection by IPC in early ischaemia reperfusion induced liver injury. The potential clinical application of the IPC effect is during liver surgery for tumors and during liver transplantation if the lobar rat data correlates with global human data. Although the technique of IPC is simple and easily applicable there would be concerns such as the increased operative time (due to period of time involving brief ischaemia and reperfusion) which may not be tolerated well in many operating theatres. Also other concerns such as, what is the ideal preconditioning time?, could IPC exacerbate damage in diseased liver (such as post chemotherapy, hepatic artery thrombosis, shock states), these questions largely remained answered since critical ischaemia times for the liver particularly with the trauma o f surgery and disease are not known. Perhaps a better option is to use IPC to evaluate the mechanism

and allow the development of pharmacological manipulation for targeted drug

intervention. Although it may be debatable whether preconditioning will ever fulfill its clinical expectations, the hope that a new therapeutic modality may emerge from this fascinating phenomenon has given great impetus to the search for its underlying mechanism. Clearly, identifying the mechanism o f IPC will allow development of pharmaceutical agents that conduct the IPC réponse. Such agents will vastly assist resection of liver for tumors and preservation of livers for transplantation. The overall conclusions of this study suggest a central role for NO in the IPC cascade. Further mechanistically descriptive studies would allow the development of therapeutic

regimens involving NO e.g., regime for NO donors administration prior to hepatectomy or liver preservation. The disadvantage of this strategy is clearly the diverse functions of NO and that NO donors may have many unwanted systemic side effects. There are no clinical studies on therapeutic evaluation using NO donors at present but it should be realized that it just might turn out to be the case where it may be better to stick with

simple and reproducible technique of IPC.

This study has shown that liver eNOS is upregulated during the IPC effect. There are many questions which require further investigation. Firstly, this study has addressed effects in early IRI (2 hour reperfusion period). Since the effect o f eNOS is short lasting, how will NO contribute if at all, to the protection in late IRI? It is therefore likely that if NO is responsible for late preconditioning in liver then the source of NO is likely to be iNOS derived. Since the induction o f iNOS takes upto 4 to 6 hours it

could be that iNOS is important in the late phase of IRI and this would fit in with the time course of late IRI. Further studies require to analyse iNOS and eNOS expression in late IRI. Survival models would further clarify this issue. In the heart there is increasing evidence for role o f NO in the late phase o f preconditioning.

The other question that will require further investigation is if there is any evidence that NOS induction with IPC is a liver alone effect. Could this be systemic NOS induction secondary to IPC? Biopsy from other organs at both early and late phase including contralateral liver biopsy would clarify whether the induction is a direct or indirect effect of IPC and whether there is systemic induction of NOS.

Another important avenue is the long term effect of IPC on microvascular perfusion. This study demonstrated that IPC caused only a limited or delayed microcirculatory damage in early IRI. Also, there was only a partial recovery of oxygenation parameters and microcirculation at the end of the 2 hrs reperfusion phase. Experimental studies by other researchers using intravital microscopy in rat liver IR have shown incomplete recovery o f HM 24 hrs after prolonged ischaemia Therefore the question that remains unanswered is whether the protective effect o f IPC is continued over several hours and days. IPC is associated with improved survival in animals. However the link between IPC and the improved survival remains unexplained. Although whether these explanations would ever be discovered remains debatable, but would be of enormous value in predicting patient outcome in the clinical situation. Recovery models after IR

looking at liver oxygenation and microcirculation and, NO activity would therefore be an interesting study for the future. The results of such a study would directly influence the potential clinical application of IPC. Another limitation o f the present study was the direct measurement of HM. Although this study showed that IPC modulated HM, it would be of new interest to elucidate why the microcirculation is altered by IPC and which componenets of the HM are modulated by IPC. Experimental studies using intravital or confocal microscopy would allow vessel measurements and assessment of various perfusion parameters including sinusoidal perfusion, leukocyte adhesion, capillary permeability and shunting. This would clarify the effect of IPC on HM and help in development o f drug targeting strategies. Recently developed physio-

pharmacological models such as the dual-perfused rat liver may allow assessment of the overall HM in finite detail to evaluate the role of NO following IRI and IPC.

There is also evidence that human liver tissue can be preconditioned. To date, in the English literature, one study has demonstrated clinical benefits o f hepatic IPC in humans Research in liver IPC is now 10 years old and although little is known about the mechanisms involved, it would be widely accepted that there is now enough animal data to justify conducting clinical research into this phenomenon.More human studies alone will prove unequivocally that IPC exists in human liver.

Three aspects can be distinguished in the process of preconditioning. The initial trigger (1), that is included in the short periods o f ischemia and reperfusion, activates signalling

pathways (2), which in turn act upon an end-effector inducing the delay of lethal ischemic damage during sustained ischemia (3). To mechanistically define the causal relationship between these processes and NO, studies could be done using genetic knockout models and, eNOS and iNOS deficient models are now readily available^^^^\

Finally, although current research on the mechanisms o f preconditioning seems to diverge more and more, it is possible that all these mechanisms converge into an as yet unidentified final common pathway. This conclusion is based not so much on the negative studies and observations discussed above but on the belief that a powerful adaptive phenomenon that is induced so easily and reproducibly in so many models, laboratories, and species is almost certain to be mediated by a universal mechanism. The data from this study suggests that nitric oxide is the key factor in hepatic ischaemic preconditioning.