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In this PhD thesis, I investigate the importance of inflammatory cytokines for activation of adrenal activity, and couple this with the use of a novel microdialysis system to assess the response of the hypothalamic-pituitary-adrenal axis in children undergoing cardiac surgery. The thesis is structured in three parts. Following the recent demonstration that there is an increased sensitivity of the adrenal cortex to ACTH in adults undergoing cardiac surgery, I investigated whether this could be mediated by cytokines. In the first part of my thesis, I describe a novel co-culture model to investigate the local, immune-adrenal crosstalk and its impact on steroidogenesis. I demonstrate that in the adrenocortical cell line ATC-7 the expression of pro-inflammatory cytokines after LPS stimulation is dependent on the ratio of adrenal and immune cells, that the presence of immune cells can modulate steroidogenesis and that this interaction can be further modulated by ACTH stimulation. In the second part, I discuss the current evidence and knowledge gaps behind the practice of glucocorticoid administration in paediatric heart surgery. I also report the results of a national survey showing a considerable variation in glucocorticoid administration within and between units. The above findings set the scene for the third part of this dissertation, that reports a study protocol for the basic understanding of the HPA axis function in children undergoing paediatric heart surgery the - Peacock Study. By using our novel automated tissue microdialysis system, I successfully measured tissue cortisol and cortisone levels in children of various ages without reducing their circulating blood volume. I also report the preliminary results of the study on 36 children undergoing surgery or catheter procedures. Firstly, I demonstrate that cortisol secretion in children is secreted with a pulsatile pattern, suggesting single-point cortisol testing is obsolete. Furthermore, I found marked changes in the perioperative cortisol to cortisone ratio. Neonates had a distinct response in that they had lower cortisol to cortisone rations presumably related to differential 11 β-HSD isoenzymes activities. Also, I show that the release of CBG and cytokines is dependent on the age of the child, the type of procedure and preoperative oxygen saturation.

There is significant variation between individual patients in the magnitude and pattern of their systemic response to cardiac surgery. Poor outcomes in these patients have been associated with a dysfunctional host response. This thesis seeks to define such variability at the level of gene expression by sequential analysis of transcription before and after surgery for a low risk group of patients undergoing elective cardiac surgery and cardiopulmonary bypass (CPB) patients using expression microarray profiling. To that aim, we analysed sequential global gene expression patterns in circulating peripheral blood leukocytes. We also investigated the role of DNA sequence variation in modulating the observed changes in gene expression. This approach allowed us to identify important genetic modulators and novel biological pathways and gain new insights into the mechanisms that regulate the host response to surgery.

Surgical myocardial revascularization remains the standard of care for patients with multi-vessel coronary artery disease. A growing body of evidence indicates that systemic inflammation and myocardial oxidative stress are associated with the development of postoperative atrial fibrillation (POAF) and low cardiac output syndrome in patients undergoing cardiac surgery. Statins have been shown to exert rapid anti-inflammatory and antioxidant effects by inhibiting myocardial NOX2 oxidases and by increasing the bioavailability of nitric oxide (NO). However, whether these so-called pleiotropic effects of statins result in improved patient outcomes remains to be established. To provide further insights into the mechanisms of action and impact on clinical outcomes of peri-operative statin treatment in patients undergoing cardiac surgery, I studied the molecular mechanisms underlying the myocardial nitroso-redox balance in samples of the right atrial appendages (RAA) obtained before (PRE) and after cardiopulmonary bypass (CPB) and reperfusion (POST) and setup two double-blind randomised placebo-controlled trials: 1) STARR (Statin Treatment on Atrial Refractoriness and Reperfusion injury), which tested the effect of Atorvastatin (80 mg once daily for up to 6 days before surgery and 5 days after) on the atrial effective refractory period (AERP, over 4 post-operative days) and superoxide production in paired PRE- and POST- RAA samples from 60 patients 2) STICS (Statin Treatment In Cardiac Surgery), which assessed the effects of peri-operative treatment with Rosuvastatin (20mg od) on POAF (assessed by continuous holter ECG monitoring for 5 days postoperatively) and myocardial injury (assessed by serial troponin I measurements) in 1922 patients undergoing elective cardiac surgery. I observed that atrial superoxide production increased significantly after reperfusion due to increased mitochondrial and NOX2 oxidase activity and to uncoupling of NOS activity. NOS activity in RAA samples decreased significantly after reperfusion (by 60%), but this reduction was not prevented by BH4 supplementation (10 μM) or NOX2 inhibition. Instead, I identified increased endothelial NOS S-glutathionylation as the main mechanism responsible for NOS uncoupling after reperfusion. In STARR, atorvastatin prevented increase in RAA superoxide production, maintained the functionally coupled status of NOS and NO bioavailability after reperfusion but had no measurable effect on postoperative AERP. In STICS, treatment with rosuvastatin significantly reduced LDL-C concentration by 48 hours after surgery but had no effect on the incidence of POAF (203 (21%) of the Rosuvastatinallocated patients vs. 197 (20%) of the placebo-allocated patients) or on perioperative myocardial damage (P = 0.80). Pre-defined subgroup analyses (age, sex, prior statin use, baseline troponin concentration, duration of randomized treatment before surgery, type of cardiac surgery, and postoperative use of anti-inflammatory drugs) did not identify any category of patient who benefited from perioperative rosuvastatin treatment. Nor were there beneficial effects on any of the other in-hospital clinical outcomes that were assessed. In conclusion, cardiac surgery on CPB is associated with myocardial nitroso redox imbalance that is reversed by perioperative intensive therapy with statins. However, these effects have no beneficial effects on common in-hospital complications after elective cardiac surgery. Although the benefits of long-term statin therapy in patients requiring myocardial revascularization are well established, the work presented in this thesis does not support routine use of perioperative intensive therapy with statins for the prevention of postoperative complications in patients undergoing elective cardiac surgery.

Quality of care and outcomes from cardiac surgery have always been of primary importance to providers and the entire health care team when caring for cardiac surgical patients. Providers in cardiac surgery have had a long history of tracking procedural morbidity and mortality rates in programs nationally and internationally. Morbidity and mortality rates vary widely among hospitals. In most studies in which procedural outcomes from cardiac surgery are evaluated, the focus has been on operative morbidity and mortality and the probability of death after a cardiac surgical procedure. Less attention has been paid to predictors of perioperative complications, and sample sizes have been small in the studies done. The focus of this dissertation was identification of perioperative predictors of perioperative complications associated with cardiac surgery to identify patients who could be at risk for morbidity or mortality related to the cardiac surgical procedure. We used a large hospital registry and had large sample sizes for our studies. By identifying those patients at risk for perioperative complications, modifiable risk factors can be addressed in order to prevent untoward outcomes. The purpose of this dissertation was to identify demographic, clinical and surgical predictors of selected common perioperative complications that can lead to morbidity and mortality in the cardiac surgical patient and the contribution of these complications to length of stay in the Intensive Care Unit (ICU) and overall length of stay in the hospital after an open chest cardiac surgical procedure with cardiopulmonary bypass. By identifying predictors of perioperative complications, we can identify patients preoperatively who may be at higher risk and intervene before a complication occurs. There are three manuscripts included in this dissertation. The aim of the first paper (N = 2399) was to determine demographic, clinical and surgical predictors of perioperative packed red blood cell transfusion in coronary artery bypass graft surgery patients undergoing cardiopulmonary bypass, and to test gender-specific models. The following predictors of transfusion were included: 1) age; 2) gender; 3) previous coronary artery bypass graft surgery; 4) body surface area; 5) number of grafts; 6) hematocrit prior to surgery; 7) hypertension; 8) diabetes mellitus; 9) history of heart failure; 10) dyslipidemia; 11) cardiopulmonary bypass time; and 12) elective versus emergent surgery. In the overall sample the following predictors were identified: 1) age; 2) body surface area; 3) cardiopulmonary bypass time; 4) prior coronary artery bypass graft surgery; 5) preoperative hematocrit; and 6) elective versus emergent surgery. Older age, lower body surface area, longer cardiopulmonary bypass time, history of prior coronary artery bypass graft surgery, lower preoperative hematocrit and emergent surgery were independently predictive of transfusion. In the male only model (n = 1721) the following were predictors of transfusion: 1) preoperative hematocrit; 2) body surface area; 3) cardiopulmonary bypass time; and 4) elective versus emergent surgery. In the female only model (n = 678) the following were predictors of transfusion: 1) preoperative hematocrit; 2) cardiopulmonary bypass time; and 3) body surface area. The aim of the second paper was to determine the association of number of major perioperative complications of open-chest cardiac surgery and cardiopulmonary bypass (N = 2350) with ICU and hospital length of stay. The number of complications was predictive of a prolonged length of ICU stay and overall hospital length of stay. Major complication was considered to be neurological complications specifically stroke, renal insufficiency and failure, respiratory failure, myocardial infarction, heart failure and bleeding which led to reoperation. We showed that one complication did increase the length of stay in the ICU but when there were two or more complications the length of stay increased significantly, revealing that it is imperative to optimize the patient as much as possible before surgery when possible to avoid any potential complications. The purpose of the third paper was to (1) determine predictors (i.e. gender, age, surgical procedure, BMI, last creatinine level, chronic lung disease, number of IMA grafts used, diabetes control, history of stroke in the past, history of MI in the past, class of heart failure, hypertension, cerebrovascular disease prior to surgery, previous cardiac procedure, post- surgical complications, re-operative bleeding, and number of major complications) of ischemic and hemorrhagic stroke in patients who have undergone cardiothoracic surgery. In terms of the first aim, we conducted a binary logistic regression. The independent variables or predictors that were tested included gender, age, surgical procedure, BMI, last creatinine level, chronic lung disease, number of IMA grafts used, diabetes control, history of stroke in the past, history of MI in the past, class of heart failure, hypertension going into surgery, cerebrovascular disease prior to surgery, previous cardiac procedure, post-surgery complication, reoperation because of bleeding, and number of major complications. When looking at the individual impact of each independent variable, the results suggested that there were three significant predictors of post-surgery stroke: BMI (Wald(1) = 6.21, p = 0.01), having chronic lung disease (Wald(1) = 5.37, p = 0.02), and having diabetes control (Wald(1) = 4.82, p = 0.03). More specifically, the results indicated that (1) having a higher BMI decreased the odds of patients experiencing post-surgery stroke with a one unit increase in BMI decreasing the odds of experiencing post-surgery stroke by 9%, (2) having chronic lung disease increased the odds of patients experiencing post-surgery stroke by 2.96, and (3) taking medication for diabetes decreased the odds of having a stroke by 0.69% or 69%. This dissertation has fulfilled and important gap in the evidence base for complications related to cardiac surgery in patients by identifying complications that could lead to a prolonged length of stay as well as predictors of stroke and the use of blood transfusion. The results of these studies can lead to the prevention of these complications and therefore the reduction in perioperative morbidity and mortality. The findings from this dissertation provide further evidence of the value of identifying patients at higher risk of complication undergoing cardiac surgery.

Background: Acute kidney injury (AKI) is a common complication in infants after heart surgery with cardiopulmonary bypass. Studies demonstrate that patients with AKI have worse clinical outcomes, including prolonged ventilation times and length of stay, likely secondary to fluid overload from oliguria. Studies suggest that early peritoneal dialysis (PD) may be superior management to diuretic medications (furosemide) for oliguria; however, this has not been proven. We aim to determine if early PD improves clinical outcomes compared to furosemide in infants with oliguria after cardiac surgery. We hypothesize that early initiation of PD will improve outcomes. Methods: This was the pilot study for a fully powered single-center randomized clinical trial. Infants with PD catheters placed during bypass were included. If infants demonstrated oliguria within the first postoperative day, they were randomized to either early PD use or furosemide. Clinical and laboratory data were collected and compared between groups. Power calculations were performed using the primary outcome of fluid balance.Results: Data were collected on the first 20 patients who met criteria for randomization; 10 to each arm. Patients in the PD arm had higher likelihood of a negative fluid balance on postoperative day 1 (60 vs 90%), demonstrated negative fluid balance 8 hours earlier (16 vs. 24 hours), were extubated 3 days earlier (3 vs. 6 days), and were transferred from the intensive care unit and hospital 6 days earlier (7 vs. 13 days; 10 vs. 16.5 days) than patients treated with furosemide. No significant adverse outcomes were reported. Using the primary outcome of negative fluid balance on postoperative day 1, a sample size of 32 participants per group was calculated for study completion. Conclusions: The use of peritoneal dialysis for patients with oliguria after heart surgery shows trends towards earlier negative fluid balance, and shorter duration of ventilation and CICU stays. Adverse outcomes were not significant in this limited review. Completion of the fully powered study is necessary to demonstrate causation of outcomes with the use of PD.

Sudden cardiac arrest is a leading cause of death in the United States and is responsible for more than 300,000 deaths every year. For the last fifty years, the guidelines of cardiopulmonary resuscitation (CPR) have been the standards to revive cardiac arrest patients. However, the major components of CPR have not changed since its birth and the success rate of CPR remains unsatisfied (5-18%). My work presented in the dissertation mainly focuses on the role of oxygen during the resuscitation and post-resuscitation periods as well as correspondent mitochondrial functions. There are four parts in the dissertation. In chapter 2, a rat CPR model was used to demonstrate that oxygen is critical during resuscitation in order to achieve return of spontaneous circulation (ROSC). Additionally, there was no significant difference between the treatment groups (21% oxygen vs. 100% oxygen resuscitation) in terms of survival rates or neurological outcome. In chapter 3, mitochondrial functions were examined in the hearts undergoing 15 minutes of cardiac arrest and 10 minutes of CPR as compared to the hearts undergoing 25 minutes of cardiac arrest. The results suggest that, despite the inherent low oxygen delivery, CPR after prolonged cardiac arrest preserves heart mitochondrial function and morphology. It demonstrates the beneficial effects of CPR from a subcellular perspective for the first time. Preservation of mitochondrial function is reflected by improved mitochondrial respiration, electron transport chain (ETC) complex activities, and preservation of the ETC proteins. Meanwhile, complex I is the most sensitive ETC complex to ischemic injury and its activity is positively correlated with mitochondrial respiration. In chapter 4, a rat cardiopulmonary bypass (CPB) model was done to study the effects of hyperoxygenation during the post-resuscitation period. After 25 minutes of normothermic cardiac arrest, hyperoxic reperfusion for one hour with CPB did show advantages compared to normoxic reperfusion in some aspects, including better systemic hemodynamics, less lactic acidosis and a small but significantly better cardiac relaxation. In addition, the benefits were also demonstrated by a better mitochondrial respiratory function. Finally, in chapter 5, a simplified method was developed to concentrate mitochondrial membrane complexes. It is a modified 2-D blue native/blue native PAGE (BN/BN-PAGE) which can be easily done with a mini-gel apparatus. Once completed, the concentrated protein complex in the gel strip is ready for SDS-PAGE or proteomic studies. Unwanted loss of protein complex is minimized, which is critical whenever the amount of mitochondrial sample is limited.

Extracorporeal circulation damages blood and causes harmful side effects such as stroke and/or systemic inflammatory response in patients. Reactions of blood components to extracorporeal circulation include complement and inflammatory reactions, coagulation and thrombogenesis, frank hemolysis, and platelet activation and adhesion to the extracorporeal circuit. Non-physiologic pressure and flow produced by blood pumps contribute to blood injury. Two pump types, roller and centrifugal, are used for maintaining flow, with various models available from different manufacturers. This study compared the effects of these two pumps in identical, isolated, artificial circuits to a non-pumped control for a period of 24 hours on heparinized porcine blood. Hematology parameters were used to evaluate blood damage. Mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, platelet count, and mean platelet volume were affected by time of circulation. Mean corpuscular hemoglobin, platelet count, and red cell distribution width were different between circulated and non-circulated blood, however no differences were found between the pumping systems in any parameter. Red blood cell count, total hemoglobin, and hematocrit were not affected by time or treatment. The changes observed in this study have implications for the use of extracorporeal circulation in the clinical setting and in future use of blood as a potential organ perfusion medium.

Polyvinyl chloride (PVC) is a brittle amorphous polymer, but processing the PVC with plasticizer results in a flexible material able to form non-rigid tubing. Plasticized PVC (pPVC) is available in medical grade products for use in extracorporeal drug and fluid delivery. There are known complications including plasticizer loss, drug interactions, spallation and raceway rupture associated with this product when used in the roller pump of the cardiopulmonary bypass circuit.This is pioneering research that studies tubing fatigued by use in the roller head raceway. The hypothesis states pPVC tubing exposed to the roller head raceway in a system simulating cardiopulmonary bypass changes material properties for tensile strength, elastic modulus and elongation. An additional hypothesis states spallation is recognized continually during compression in the roller head raceway and occurs at multiple surfaces of the tubing inner lumen. Better evaluation of polymers approved and chosen for the specific medical application of extracorporeal circulation by the roller head raceway and other approved devices will be obtained through this research protocol. With this basic knowledge clinicians will better understand the product selections available and better understand how to evaluate material offered from the manufacturers for specific applications.Minimal research recognizes the basic science of spallation and catastrophic rupture that occur during cardiopulmonary bypass. Cardiopulmonary bypass material selection was made to alleviate these concerns including the switch to pPVC from other non-rigid polymers, such as silicone and natural rubber. PVC is known to have a complicated morphology unstable during the compression in the roller head raceway. The initial phase of the study will trend the change in mechanical behavior of the polymer tubing after exposure to fatigue stresses of cyclic bending and compression by the roller head contact. This includes mimicking set-up and priming techniques used to prepare the equipment for patient care. Physiological saline and lipid will be used to duplicate the blood interaction with the pPVC known to influence material chemical stability. Pump settings for occlusion and RPM will simulate clinical cardiopulmonary bypass protocols. The material takes on a more crystalline structure leading to changes in the performance of the pPVC material. This simulation will develop the knowledge to understand the changes in behavior experienced during a typical cardiac surgical procedure.

It has been suggested that desmopressin acetate (DDAVP) administration reduces blood loss after cardiac surgery. We have investigated the effect of DDAVP administration in a double-blind, randomized, prospective trial including 100 patients placed on cardiopulmonary bypass during surgery. Fifty patients received 0.3 micrograms/kg DDAVP and 50 patients received a placebo administered in a 50 ml saline solution over 15 min when cardiopulmonary bypass had been concluded. Results showed no significant differences either in total blood loss per square meter (458 +/- 206 ml in the DDAVP group vs 536 +/- 304 ml in the placebo group) or in necessity for red cell transfusions (1642 +/- 705 ml in the DDAVP group vs 1574 +/- 645 ml in the placebo group) in the first 72 hr after surgery. Only intraoperative blood loss per square meter was significantly lower (p less than .02) in the DDAVP group (131 +/- 106 ml) as compared with the placebo group (193 +/- 137 ml). The prolongation of bleeding time and the decrease of factor VIII:C and factor VIII:von Willebrand factor 90 min after treatment were significantly lower (p less than .001) in the DDAVP group as compared with the placebo group. We conclude that the administration of DDAVP in patients placed on cardiopulmonary bypass during surgery does not reduce total blood loss and is only effective in reducing intraoperative bleeding.

BACKGROUND: Patients on cardiopulmonary bypass (CPB) have an increased susceptibility to postoperative bleeding. Previous reports using desmopressin acetate (DDAVP) for the prevention of postoperative bleeding have given contradictory results, whereas the protease inhibitor aprotinin has been shown to reduce blood loss after this type of surgery. This randomized study was performed to assess the efficacy of DDAVP versus aprotinin in the prevention of bleeding after CPB. METHODS AND RESULTS: One hundred nine of 122 eligible patients were randomized to four different groups: Group A (n = 28) received aprotinin starting with a bolus of 2 x 10(6) KIU followed by a continuous infusion of 0.5 x 10(6) KIU/h until the end of surgery; group B (n = 25) received of DDAVP 0.3 micrograms/kg i.v. on completion of CPB; group C (n = 28) received two doses of DDAVP, the first as in group B and an additional dose 6 hours after surgery; group D (n = 28) received no treatment. There was a marked reduction of postoperative blood loss either at 12 hours (P < .01) or 72 hours (P < .02) in the aprotinin group compared with all other groups, whereas no significant effect was observed in either of the two DDAVP regimens. A significant reduction in the amount of blood used was observed only in the aprotinin group (P < .01). Of the plasma fibrinolytic components assayed, there was a significant reduction of the fibrin degradation product generation in the aprotinin group (P < .001), whereas a significant systemic hyperfibrinolysis was observed in both DDAVP-treated groups and the control group. No side effects related to the study drugs were observed in any patient. CONCLUSIONS: Aprotinin inhibited fibrinolysis; this correlated with a significant reduction of postoperative blood loss and need for blood replacement after CPB. Neither one nor two doses of DDAVP had a beneficial effect. Aprotinin offers a better alternative than DDAVP in the prevention of bleeding after CPB.

The thoracic approach for cardiac surgery in a patient with a tracheostoma can result in difficult problems, such as mediastinitis, stoma necrosis or inadequate operative exposure. We present a distinct approach consisting of an incision at the second intercostal space, transverse sternum transection and longitudinal median sternotomy to the xiphoid process, performed for coronary artery bypass grafting and aortic valve replacement, in a patient with previous tracheotomy. This approach permitted adequate surgical exposure for cardiopulmonary bypass, aortic valve replacement and coronary revascularization procedures.