Effect of remote ischaemic preconditioning in cardiac dysfunction and end-organ injury following cardiac surgery with cardiopulmonary bypass in children: A translational approach investigating clinical outcome and myocardial molecular biology

Congenital heart disease (CHD) is the most common birth defect, causing an important rate of morbidity and mortality. Treatment of CHD requires surgical correction in a significant percentage of cases which exposes patients to cardiac and end organ injury. Cardiac surgical procedures often require the utilisation of cardiopulmonary bypass (CPB), a system that replaces heart and lungs function by diverting circulation into an external circuit. The use of CPB can initiate potent inflammatory responses, in addition a proportion of procedures require a period of aortic cross clamp during which the heart is rendered ischaemic and is exposed to injury. High O2 concentrations are used during cardiac procedures and when circulation is re-established to the heart which had adjusted metabolically to ischaemia, further injury is caused in a process known as ischaemic reperfusion injury (IRI). Several strategies are in place in order to protect the heart during surgery, however injury is still caused, having detrimental effects in patients at short and long term. Remote ischaemic preconditioning (RIPC) is a technique proposed as a potential cardioprotective measure. It consists of exposing a remote tissue bed to brief episodes of ischaemia prior to surgery in order to activate protective pathways that would act during CPB, ischaemia and reperfusion. This study aimed to assess RIPC in paediatric patients requiring CHD surgical correction with a translational approach, integrating clinical outcome, marker analysis, cardiac function parameters and molecular mechanisms within the cardiac tissue. A prospective, single blinded, randomized, controlled trial was conducted applying a RIPC protocol to randomised patients through episodes of limb ischaemia on the day before surgery which was repeated right before the surgery started, after anaesthesia induction. Blood samples were obtained before surgery and at three post-operative time points from venous lines, additional pre and post-bypass blood samples were obtained from the right atrium. Myocardial tissue was resected during the ischaemic period of surgery. Echocardiographic images were obtained before the surgery started after anaesthetic induction and the day after surgery, images were stored for later off line analysis. PICU surveillance data was collected including ventilation parameters, inotrope use, standard laboratory analysis and six hourly blood gas analysis. Pre and post-operative quantitation of markers in blood specimens included cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP), inflammatory mediators including interleukins IL-6, IL-8, IL-10, tumour necrosis factor (TNF-α), and the adhesion molecules ICAM-1 and VCAM-1; the renal marker Cystatin C and the cardiovascular markers asymmetric dymethylarginine (ADMA) and symmetric dymethylarginine (SDMA). Nitric oxide (NO) metabolites and cyclic guanosine monophosphate (cGMP) were measured before and after bypass. Myocardial tissue was processed at baseline and after incubation at hyperoxic concentration during four hours in order to mimic surgical conditions. Expression of genes involved in IRI and RIPC pathways was analysed including heat shock proteins (HSPs), toll like receptors (TLRs), transcription factors nuclear factor κ-B (NF- κ-B) and hypoxia inducible factor 1 (HIF-1). The participation of hydrogen sulfide enzymatic genes, apelin and its receptor were explored. There was no significant difference according to group allocation in any of the echocardiographic parameters. There was a tendency for higher cTnI values and inotropic score in control patients post-operatively, however this was not statistically significant. BNP presented no significant difference according to group allocation. Inflammatory parameters tended to be higher in the control group, however only TNF- α was significantly higher. There was no difference in levels of Cystatin C, NO metabolites, cGMP, ADMA or SDMA. RIPC patients required shorter PICU stay, all other clinical and laboratory analysis presented no difference related to the intervention. Gene expression analysis revealed interesting patterns before and after incubation. HSP-60 presented a lower expression at baseline in tissue corresponding to RIPC patients, no other differences were found. This study provided with valuable descriptive information on previously known and newly explored parameters in the study population. Demographic characteristics and the presence of cyanosis before surgery influenced patterns of activity in several parameters, numerous indicators were linked to the degree of injury suffered by the myocardium. RIPC did not reduce markers of cardiac injury or improved echocardiographic parameters and it did not have an effect on end organ function; some effects were seen in inflammatory responses and gene expression analysis. Nevertheless, an important clinical outcome indicator, PICU length of stay was reduced suggesting benefit from the intervention. Larger studies with more statistical power could determine if the tendency of lower injury and inflammatory markers linked to RIPC is real. The present results mostly support findings of larger multicentre trials which have reported no cardiac benefit from RIPC in paediatric cardiac surgery.

An investigation into the role of interleukin-6 in linking systemic and local inflammatory responses in patients with colorectal cancer

Colorectal cancer is the second most common cause of cancer death in the UK. It is accepted that both tumour and host factors are important determinants of disease progression and survival. While systemic and local inflammatory responses are increasingly recognized to be of particular importance the understanding of the mechanisms linking these important inflammatory processes remains unclear. This thesis examines the prognostic importance of measures of systemic and local inflammation and proposes a hypothesis for a link between tumour necrosis, systemic and local inflammatory responses in patients with colorectal cancer. Chapter 3 reports the comparison of the prognostic value of longitudinal measurements of systemic inflammation in patients undergoing curative resection of colorectal cancer. In Chapter 3 the results demonstrate that there was no significant overall change in either mGPS or NLR from pre- to post- operatively. This study highlighted the associations between pre- and post- operative mGPS and NLR and T-stage (p<0.001), TNM stage (p<0.005) and cancer-specific survival. The relationships between pre-operative measurements were examined using multivariate analysis. For pre-operative measurement both mGPS and NLR were associated with cancer-specific survival while when post-operative measures were examined only mGPS was specifically associated with cancer-specific survival (HR 4.81, CI 2.13-10.83, P<0.001). Chapter 4 examines the prognostic value of the Klintrup-Makinen scoring method and the existing limitations with regard to its clinical utility. An automated scoring method using commercially available image analysis software was developed and compared with manual scoring of tumour inflammatory infiltrates. This study demonstrated that both manual K-M scoring (p<0.001) and automated K-M scoring (p<0.05) had prognostic value in patients who had undergone potentially curative resection of colorectal cancer, and that the novel automated method may provide an objective method of assessment of tumour inflammatory infiltrates using routinely stained haematoxylin and eosin sections of tumour samples. In chapter 5 a hypothesis was proposed that Interleukin-6 may link tumour necrosis and systemic and local inflammatory responses in patients with colorectal cancer. This chapter examined the basis for this hypothesis, which is presented in figure 5.1. In addition, in chapter 5 the importance of this potential link is examined. In chapter 6, the hypothesis outlined in chapter 5 was examined in a cohort of patients who had undergone attempted curative resection of colorectal cancer. This study examined the inter-relationships between circulating mediators, in particular IL-6, tumour necrosis and systemic and local inflammatory responses. This results of this study demonstrated that IL-6 was associated with tumour necrosis (<0.001) and mGPS (<0.001) independent of T-stage. Thus adding weight to the hypothesis that elevated circulating concentrations of IL-6 may play a role in modulating both the systemic and local inflammatory responses in patients with cancer. Chapter 7 further develops the hypothesis that IL-6 signalling may be important in modulating systemic and local inflammatory responses in patients with colorectal cancer. Further, in chapter 7 the basis for the role of trans-signalling in this signaling pathway was examined. In this study, we reported that neither expression of the soluble IL-6 receptor or soluble gp130 were associated with systemic or local inflammatory responses. As a result the possible reasons for these findings were explored and future work suggested. A prospective database of patients undergoing attempted curative resection of colorectal cancer in Glasgow Royal Infirmary was used throughout this thesis. This database was created and is maintained regularly by successive research fellows at the Royal Infirmary. The work presented in this thesis highlights the importance of the host response in the form of systemic and local inflammation in patients with colorectal cancer and proposes a link between these responses and tumour necrosis. In addition, this work adds weight to the body of evidence suggesting that assessment of these host responses may improve stratification to treatment for patients with colorectal cancer. Further, this work proposes a mechanistic link, between tumour necrosis, systemic and local inflammatory responses through Interleukin-6, that merits further investigation.