Title: Effects of new methods in cardiac surgery, ARRS J7-7138

Type of reasearch: Basic scientific project

Founding: Slovenian Research Agency, ARRS

Bleiweisova cesta 30, 1000 Ljubljana

www.arrs.gov

Duration: 2016, 2017, 2018

Colaboration: University Medical Centre Ljubljana, Slovenia

University of Ljubljana, Medical Faculty, Ljubljana, Slovenia

Head: Prof. Matej Podbregar, e-mail: matej.podbregar@kclj.si

Ethical approval: National Medical Etics Committee, Republic Slovenia, http://www.kme-nmec.si/

International registration: ClinicalTrials.gov Identifier: NCT02666703 NCT02697786

Abstract

Background: Although cardiopulmonary bypass (CPB) is indispensable for most open heart surgery, its use is associated to unwanted systemic inflammatory response. This complex chain of events has many similarities with sepsis and may contribute to the development of postoperative complications, all the way to multiple organ failure and death. It was shown that CPB can also trigger an anti-inflammatory response. The balance of inflammatory and anti-inflammatory mediators determines inflammatory response and clinical outcome. Considerable efforts have been focused on therapeutic interventions to reduce the inflammatory reaction related to CPB, including pharmacological strategies, i.e.use of corticosteroids, and improvment in surgical techics or mechanical devices. Such therapies may have positive effects on outcome of patients after open heart surgery. Serum paroksonaza 1 (PON1) is a lipo-laktonaza being associated with HDL, which has anti-inflammatory role. Exosomes are nanovesicles released from cells and are mediators of proximal as well as distant cell-to-cell signaling. The question is, what is clinical importance of non-selective artificial imunoadsorbtion during CPB, focusing specially on PON1 and exosomes. Brain injury after aortic valve replacement (AVR) includes stroke, coma, encephalopathy, delirium and cognitive decline. Intraoperative cerebral embolization appears to be an important mechanism of such injury, as gaseous or solid cerebral emboli can cause ischemia, inflammation and edema, consequently causing cerebral microinfarctions. AVR by way of a full sternotomy (FS) is the standard approach in the treatment of aortic valve disease. Another approach is when surgeon performes AVR through partial sternotomy, this is minimally invasive surgery (MIS). Compared with conventional surgery (FS), MIS has been shown to reduce postoperative mortality, morbidity, and pain while providing faster recovery, a shorter hospital stay, and better cosmetic results. However, due to technically more demanding procedure, MIS may lead to prolonged CPB time and incomplete aorta de-airing with an increased risk for cerebral gas embolization. Therefore, the choice of MIS might bear an augmented risk for brain

injury. Cerebral microembolic signals (MES) may be detected in real time with transcranial Doppler ultrasound (TCD). Research objectives: Our aim is to improve cardiosurgical techniques, to minimaze proinflamatory

response after CPB and decrease the decline of cognitive functions. The purpose of research,

conducted at the Clinical Department of Cardiovascular Surgery at the University Medical Centre

Ljubljana, is to analyze the effect of selective in vitro imunoadsobtion on development of

inflammatory immune to answer and of protective mechanisms of CPB in cardiac surgery.The second

aim is comparison of two surgical techniques for AVR. We will specially focus on intraoperative MES,

level of S100B protein (marker of brain injury), postoperative cerebrovascular reactivity and cognitive

function.

Detailed description of the work programme

The primary purpose of the proposed project is to improve treatment of patients during and after cardio-vascular surgery. The project is divided into two partially linked work packages (WP). The first WP (WP1) will focus on the impact of new methods on immunological status of patients during and after cardio-pulmonary bypass (CPB), studying especially effects of adsorbtion therapy on pro- / anti-inflammatory mediators and on other protective mechanisms. We also want to evaluate inflammatory and anti-inflammatory response and cellular immunity in patients operated with CBP, who will receive corticosteroid compared to placebo during surgery. The main purpose is to limit the cytokine storm (Systemic Inflammatory Response Syndrome, SIRS) between CBP and consequently to reduce the anti-inflammatory reaction-immune paralysis (Compensatory Anti-inflammatory Response Syndrome, CARS), which is associated with secondary infections and increased mortality of patients. In the second WP (WP2) we want to investigate neurological disorders and brain injury during aortic valve replacement (AVR) via a minimally invasive surgery (MIS) approach, and compare it with standard surgical techniques-full sternotomy (FS). We will focus mainly on the extent of the brain micro embolisms, vascular reactivity and a decline in cognitive function. We want to find out if MIS is equivalent approach comparing to FS in demanding surgical procedures such as AVR, which requires CPB regarding the gaseous microembolism. WP1 Effects of extracorporeal cytokine adsorbtion on immune response and immune status after cardio-pulmonary bypass Background CPB is associated with SIRS and so-called cytokine storm, which leads to an excessive inflammatory response , followed by a powerful anti- inflammatory response, CARS. SIRS and CARS are consequently important causes for the hemodynamic and organic dysfunction, organ failure, secondary infection, and even death of the patient. The PON1 enzyme confers protective role in response to inflammation. Since the size of PON1 (10kD - 50kD) is in the range of molecular weight of molecules, which are adsorbed by extracorporeal adsorption, its fate during adsorption is unclear. Aim To determine effect of extracorporeal cytokine adsorbtion on immune response and immune status and protective mechanisms of the patients undergoing complex cardiac procedure with CPB. Specific aims in WP1 are two: WP 1.1. Determination the effects of extracorporeal adsorbtion therapy CytoSorb® capsula (CytoSorbents Medical Inc.,New Jersey, ZDA) on post-operative immune status and other protective mechanisms in patients undergoing complex cardiovascular procedures under CPB WP 1.2. Comparison of postoperative immunological status and protection mechanisms in patients undergoing complex cardiac-vascular procedures with CPB, without the use of CytoSorb®; with or without the use of methylprednisolone during CPB. Relevance. By reducing the SIRS during and after CPB by extracorporeal adsorbtion of cytokines, reduced level of the inflammatory cytokine storm, we expect improvement of the hemodynamic stability of patients, reduced need for inotropic and vasoactive therapy, reduced need for volume therapy, all of which leads to less frequent dysfunctions and organ failure, i.e. less acute renal failure. The anti- inflammatory response, which is a reaction to the inflammatory response, is expected to be less expressed after adsorbtion of cytokines between CPB, which may be associated with less secondary infections. In comparison with the extracorporeal body immuno-adsorption corticosteroid, use in priming fluid of CPB, are unlikely to sufficient limit the inflammatory response, but only to amplify the anti-inflammatory response, which is associated with a greater incidence of secondary infections. With the findings of our project could lead us to modify the protocol of CPB to reduce cytokine storm and consequently alleviate immune paralysis of our patients and improve their outcomes. In the future it could be possible to modulate the inflammatory response with continous infusion of PON1 in the cardiac surgery patients. Patients , who will be invited in the study. Patients, who will be invited in the study, will be selected from patients at the Clinical Department of Cardiovascular Surgery, University Medical Centre in Ljubljana, who are intended for the planned and complex heart operation using the CPB. Study was approved by Medical Ethics Committee of the Republic of Slovania (no.: 118/02/15 , dated 20.02.2015 ). We declare that no invitations will be accompanied by pressure or improper solicitation. Participation in all respects a patient's voluntary and it will not receive refunds. At the beginning of the study, each of the patients will be informed about the progress of research in a simple manner. They will be presented a written consent for surgery and anesthesia and consent to participate in the study; patients will be given written information about the study. Patients and methods In the study 60 patients will be included, admitted for elective, complex heart surgery with the use of CPB . Inclusion criteria:

1. Elective cardiac surgery with an expected duration of CPB more than 120 minutes (i.e. combined valve and coronary bypass grafting surgery, operative interventions on several valves, surgery of the ascending aorta and aortic arch, as well as re-operations of the same type)

2. Age > 18 years

Exclusion criteria:

1. Disagreement to participate in the study 2. Age < 18 years 3. Pregnant women 4. Emergency procedures 5. Heart transplantation 6. Implantation of mechanical circulatory support (i.e. LVAD (left ventricular assist device), RVAD (right ventricular

assist device) or TAH (total artificial heart)) 7. Treatment with chemotherapy, immunosuppressive therapy 8. Treatment with anti-leukocyte drugs or TNF-alpha blockers 9. Immunocompromised patients (AIDS), leucopenia (< 4,0x109 / L) 10. Clinical and/or laboratory signs of infection (CRP >2 mg/dl 9/L) 11. Serum creatinine >2 mg/dl (176 micmol/L) 12. Bilirubin >2 mg/dl (34.2 micmol/L) 13. History of stroke 14. Malnourished patients, BMI < 18

Before anesthesia, patients will be randomized into three groups using the system of random numbers:

1. Study group – n=20 patients undergoing cardiac procedures with prolonged CPB using CytoSorb capsule during CPB (CytoSorb, CytoSorbents Inc., New Jersey, USA)

2. Control group – n=20 patients undergoing cardiac procedures with prolonged CPB, without the use of CytoSorb capsule during CPB

3. Corticosteroid group – n=20 patients undergoing cardiac procedures with prolonged CPB, without using CytoSorb capsule, but with methylprednisolone (Solumedrol 1 g) in the CPB priming solution.

Preoperative assessment, intraoperative course and early postoperative care will be according to the standard operative guidelines (SOP, Standardni Operativni Postopki), that were accepted at the Clinical department of anesthesia and perioperative intensive therapy, University medical centre Ljubljana, in 2009 and re-validated in 2013. General preoperative assessment will include: medical history, clinical examination, ECG, chest X-ray, echocardiography, heart catheterization, pulmonary functional tests and laboratory tests: blood count with leukocyte formula and rate of sedimentation; biochemical tests (blood glucose, electrolytes, BUN, creatinine, tests of hepatic function, protein status, coagulation status, biomarkers of infection: C-reactive protein (CRP), procalcitonin (PCT)). All examinations will be performed before surgery; laboratory test will be repeated after surgery, after 24hours, after 48 hours and on the 5th day after surgery, as part of standard protocolled assessment at our institution. We will familiarize with patient's medications and decide which of them patients will take until the day of surgery, considering their condition and accepted guidelines. Anesthesiological procedure After preoperative assessment and premedication with benzodiazepines, patients will be operated in general anesthesia (GA). Before introduction of general anesthesia, in each patient an arterial cannula will be inserted for invasive measurement of arterial blood pressure. Induction of GA will be intravenous, using fentanyl 5- -0,3 mg/kg and rocuronium bromide 0,6 mg/kg for tracheal intubation. Patients will be ventilated with volume or pressure controlled ventilation with tidal volume (Vt 6-8 ml/kg ideal body weight), with the intention to maintain end tidal pCO2 (EtCO2) between 4 and 5 kPa. After introduction of GA, in each patient a central venous catheter will be inserted (PreSep Central Venous Oximetry catheter, Edwards, USA) for hemodynamic measurements, as well as MAC (multi-access catheter) for fluid replacement therapy. GA before CPB will be maintained with volatile anesthetics and continuous infusion of opioid analgesic remifentanyl; for maintenance of GA during CPB we will use continuous infusions of remifentanyl and intravenous anesthetic propofol (4-12 mg/kg/h). Standard and extended hemodynamic monitoring will be used during general anesthesia: ECG, invasive arterial blood pressure, central venous pressure, cardiac output, systemic vascular resistance, central venous oxygen saturation, pulse pressure variation, stroke volume variation (EV 1000 platform, Edwards, USA). We will also monitor: hemoglobin oxygen saturation, capnography, cerebral oxygen saturation (NIRS, near infrared spectroscopy), depth of anesthesia (BIS, bispectral index), body temperature, urine output, acid base status with blood gas analysis. We will note type and duration of surgery, duration of CPB, aortic cross-clamping time, blood loss and quantity of crystalloids, colloids and blood components given,

use of inotropic and/or vasoactive drugs, use of insulin. During every procedure we will use transoesophageal echocardiography to assess global cardiac function, regional wall motion abnormalities, valvular function, volume status, as well as for de-aeration before unclamping of the aorta. Type of CPB that we’ll use is standard (type of oxygenator - Inspire (Sorino, Milan, Italy) or Fusion (Medtronic, Minneapolis, USA). Composition of priming solution: ringer, 20% mannitol, heparin. In the study group of patients where CytoSorb capsule will be used during CPB, it will be installed on the CPB machine in a parallel circuit. The flow through through the capsule (filter) will be driven by a roller pump with 200 ml/min. Following surgery, patients will be transferred in cardiac-vascular intensive care unit (CV-ICU) intubated, sedated and mechanically ventilated. They will be awaken and extubated when extubation criteria are fulfilled: awake, cooperative patient, with completely reversed neuro-muscular function, hemoglobin oxygen saturation above 96% with FiO2 0,5 or less, EtCO2 less than 6 kPa, stable hemodynamic and normal core temperature, with retrosternal drainage less than 100 ml/h. Postoperative pain will be treated with continuous intravenous infusion of opioid analgesic morphine and nonsteroidal analgesic metamizol mixture. Measurements: We will document demographic characteristics of patients, their preoperative medical status, as well as intraoperative data (type and duration of surgery, duration of CPB, period of ischemia, hemodynamic measurements, usage of inotropic/vasoactive therapy, insulin, fluids, blood and blood components); duration of mechanical ventilation in CV-ICU, duration of intensive therapy, postoperative complications, 30-day mortality. For the purpose of the study, postoperative hemodynamic values will be recorded in the first 5 postoperative days. Determination of biochemical markers. Blood samples will be obtained:

1) Before induction of general anesthesia 2) After completion of CPB 3) After completion of surgery (i.e., on admission in CV-ICU) 4) 24 hours after surgery 5) 48 hours after surgery 6) On 5th postoperative day

In the study group using CytoSorb capsule, we will do 2 additional blood samplings – during CPB- after 120 minutes of CPB: 7) from the blood in the machine circuit before entering the capsule and 8) after leaving the capsule. The following biochemical markers will be determined:

1. Pro- and anti-inflammatory cytokines: TNF- -2, IL-6, IL-8, IL-10 in plasma ( ELISA) 2. complement C5a

3. PON1 activity, genotype and phenotype (genotype and phenotype will be determined only in the first blood sample) 4. Lipide status (HDL in LDL holesterol) 5. hs-CRP (high sensitivity CRP), PCT (ECLIA), Differential blood count 6. Albumin, fibrinogen 7. Cell based immune status:

Lymphocyte: CD3, CD4, CD8, HLA-DR, CD163,CD 64, CD16+CD56, Treg – flow cytometry Myeloperoxidase

8. Exosomes in serum 9. Classical clinical and microbiological approach will be used to determine possible infection in patient.

Postoperative complications: We will record postoperative complications such as: bleeding, hemodynamic instability, impaired respiratory function, worsening of renal, liver and brain functions. Patients will be transferred on ward when following conditions are met: hemoglobin oxygen saturation of 94% or more at FiO2 of 0.5 or less, hemodynamic stability without hemodynamically significant arrhythmias, with diuresis > 0.5 ml/kg/hour, without intravenous inotropic or vasopressor therapy, as well as without delirium or epileptic activity, without signs of infection. Follow-up by telephone will be carried out 30 days after the procedure, when we will focus on late postoperative morbidity and mortality. All personnel included in the study, together with those involved in ICU treatment, will be ‘blinded’ for the assigned treatment throughout whole duration of the study, except the anesthesiologists and perfusionists in operating theatre, who, on the other hand, will not be included in data collection, input and analysis. Expected results: In this clinical study we will test the hypothesis that selective extracorporeal immunoadsorbtion during prolonged CPB in cardiac surgery affects the postoperative immunological status and protective mechanisms, with modulation of the systemic inflammatory response and anti-inflammatory mediators. After completing the survey and statistical processing of data, we expect results will demonstrate that CytoSorb capsule adsorbs and thus reduces the concentration of cytokines and anti-inflammatory enzymes during CPB in cardiac surgery. We also expect the results of the study to show a difference in the concentration of cytokines and other inflammatory markers between patients undergoing CPB with and without corticosteroid prophylaxis. Expected results

1. Identification of CPB method which produces less pro-inflammatory response after surgery

2. Identification of CPB method which produces less immune- paralysis (anti-inflammation), which is associated with higher

rate of ICU/hospital acquired infection

3. Determination of effects of different CPB methods on PON1, which can be used as systemic anti-inflammatory agent in future. Applicability: The data we will receive after completing the study could prove the reliability of therapy with selective immune-adsorbtion in the field of cardiac surgery using extracorporeal circulation. When such results come from a prospective, controlled, blinded clinical study, immune-adsorbtion could be implemented as a routine treatment measure in heart surgery, especially because pharmacologic protective strategies may be associated with postoperative unwanted side-effects, such as increased frequency of infections, hyperglycemia, prolonged postoperative mechanical ventilation of the lungs etc. Through the simultaneous reduction in the concentration of pro- and anti-inflammatory cytokines during extracorporeal blood circulation, immune-adsorbtion could repeal deactivation of monocytes, reduce complications and improve functioning of the organ systems, as well as patient outcome. This way, postoperative morbidity and mortality in patients undergoing heart surgery is decreased. WP2 Neuroprotection during minimally invasive approach to aortic valve replacement Background MIS-AVR is associated with around 4% of cerebrovascular incidents compared to 2% with FS approach (Borger et al. Ann Thorac Surg 2015) We are interested in whether there is a link to the frequency and degree of neurological complications with technically more difficult removing air bubbles from the chambers of the heart after AVR and consequent higher incidence of emboli. In addition to micro-embolization between surgery, disturbed autoregulation is one of the basic factors of inadequate cerebral perfusion, since during surgery possible changes of systemic blood pressure outside the self-regulating areas of the brain could results in inadequate perfusion and in a lack of oxygen in the brain. Aim. We want to compare MIS - AVR with conventional FS AVR in relation to the incidence of micro-emboli, in biomarkers of brain damage and the incidence of neurological complications and decline in cognitive function. Further we would like to examine the vascular reactivity of brain vasculature in relation to the number of micro-emboli and brain damage during the operation. We also wish to explore the importance of preserved autoregulation of cerebral flow before surgery on the occurrence of neurological complications after surgery. The specific objective of the WP2 are four: 1. Determination of the number of emboli during AVR in patients operated by the classic FS technique compared with MIS 2. Determination of the relationship between the frequency of emboli with post-operative indicators of brain injury 3. Identify the link between change of vascular reactivity of brain blood vessels and the number of micro-emboli and the brain damage after surgery 4. Determine the impact of preoperative vascular reactivity to neurological complications post-operatively Relevance. By confirming the hypothesis that the brain injury during MIS – AVR is more common than in the conventional with FS approach and that it is due micro-embolization we could in the future develop additional techniques to deaeration hearts at MIS. Since cognitive dysfunction after cardiac surgery is very common, our data on vascular cerebral reactivity will provide us valuable information for the development of new diagnostic and therapeutic options for precise control of patients with disturbed autoregulation, who are probably more susceptible to neurological damage. Patients and methods This will be a prospective interventional clinical study on 60 patients undergoing AVR, divided in two groups according to the chosen surgical procedure. Group I will consist of 30 patients admitted for AVR conducted with FS. Group II will consist of 30 patients admitted for AVR conducted with MIS. Patients will be randomly allocated to different treatment groups. European System for Cardiac Operative Risk Evaluation score 2 (EuroSCORE, www.euroscore.org) will be used for assessment of risk of death after surgery. Clinically study was approved by the Medical Ethics Committee of the Republic Slovenia (no. 22k / 04/ 15, dated 04.23.2015). We declare that no invitations will be accompanied by pressure or improper solicitation. Participation in all respects a patient's voluntary and it will not receive refunds. At the beginning of the study, each of the patients in a simple manner informed about the progress of research. They will be presented a written consent for surgery and anesthesia and consent to participate in the study; patients will be given written information about the study. Inclusion criteria:

1. Isolated aortic valve stenosis as well as asymptomatic patients with depressed systolic function 2. Symptomatic patients with normal or depressed left ventricular function 3. Patients with American Society of Anesthesiologist (ASA) Physical Status Classification 2 or 3 4. Patients without present stenosis on carotid arteries or already resolved by stenting or surgery

Exclusion criteria: 1. History of brain stoke 2. EF less than 30 % 3. History of alcohol abuse

4. Epilepsy or history of psychiatric illness and antipsychotic drugs Patients who will participate in the study will be visited by anesthesiologist day before the surgery, their general condition will be assessed and mini mental test will be done to assess their cognitive function (Standardised Mini-Mental State Examination, www.ihpa.gov.au/internet/ihpa/publishing.../smmse-guidelines-v2.pdf) Visually evoked cerebral blood flow velocity response (VEFR)(Performed at Institute of physiology, Medical Faculty, University of Ljubljana, as.Fabjan A., MD PhD) Experimental trans-cranial doppler (TCD) testing will be performed one week before surgery and one week after surgery. The evaluations will be carried out in a semi-dark, acoustically isolated room with a temperature of approximately 22⁰C. Arterial blood pressure (ABP) and heart rate (HR) will be continuously monitored in the left hand with a non-invasive finger cuff, holding the finger at heart level (Finapress 2300 Blood pre, Ohmeda, USA). End-tidal CO2 (Et-CO2) levels will be monitored through a face mask using capnometer (Oscaroxy capnometer, Datex, Finland). For insonation through the temporal transcranial ultrasonic bone window, 2 MHz pulsed wave Doppler monitoring probes of the Doppler device (Delica-9 series, SMT Medical, Germany) will be mounted on the helmet, to record flow velocity in the P2 segment of the left posterior cerebral artery, and the M1 segment of the right middle cerebral artery, as described elsewhere in detail (Azevedo et al. 2010). Beat-to-beat peak systolic, mean and end diastolic blood flow velocities (BFV), ABP, Et-CO2 and stimulus marker will be digitally recorded in the Doppler device. Subjects will sit in front of the display surrounded by a uniform luminance field of 5 cd/m2. To obtain a constant pupil diameter, before VEP recording, each subject will adapt to the ambient room light for 10 min. Visual stimulus will consist of full-field checkerboard-like patterns (contrast 80%, mean luminance 250 cd/m2) generated on a LCD monitor and reversed in contrast at a rate of 2/s. Subjects will be instructed to fixate a red dot in the middle of the screen to maintain stable fixation. The visual-evoked paradigm will consist of 10 cycles, each with a resting phase of 20 s with closed eyes and a stimulating phase of 40 s of visual stimulus presentation. Changes between phases will be signaled acoustically using a tone. TCD beat-to-beat data of 10 repeated rest-stimulation phases will be interpolated linearly with a time resolution of 50 ms for averaging procedures. Data will be transformed to relative data using the resting flow velocity level averaged for a time span 5 s before the beginning of the stimulation as a baseline. The baseline will be set to zero. The transformation of visually evoked absolute flow velocity data into relative data enables independence from the insonation angle, and allows correlation of flow velocity changes to flow changes (Rosengarten et al. 2001). For functional Doppler data analysis, peak systolic flow velocities will be used because they are less prone to artifacts (Rosengarten and Kaps 2002). The evoked flow velocity responses over the 40 s stimulation period will be averaged and analyzed using a custom made software. VEFR will be determined as a relative increase in flow velocity from baseline to the average flow velocity of the last 10 s of each stimulation phase. For the analysis specially dedicated computer program will be used to compared the average change of blood flow flow between the 40s visual stimulation with unstimulated phase (Fabjan et al. 2012). Operative procedure Preoperative assessment, intraoperative course and early postoperative care will be according to the standard operative guidelines (SOP, Standardni Operativni Postopki), that were accepted at the Clinical department of anesthesia and perioperative intensive therapy, University medical centre Ljubljana, in 2009 and re-validated in 2013. General preoperative assessment will include: medical history, clinical examination, ECG, chest X-ray, echocardiography, heart catheterization, pulmonary functional tests and laboratory tests: blood count with leukocyte formula and rate of sedimentation; biochemical tests (blood glucose, electrolytes, BUN, creatinine, tests of hepatic function, protein status, coagulation status, biomarkers of infection: C-reactive protein (CRP), procalcitonin (PCT)). All examinations will be performed before surgery; laboratory test will be repeated after surgery, after 24hours, after 48 hours and on the 5th day after surgery, as part of standard protocolled assessment at our institution. We will familiarize with patient's medications and decide which of them patients will take until the day of surgery, considering their condition and accepted guidelines. Anesthesiological procedure After preoperative assessment and premedication with benzodiazepines, patients will be operated in general anesthesia (GA). Before introduction of general anesthesia, in each patient an arterial cannula will be inserted for invasive measurement of arterial blood pressure. Induction of GA will be intravenous, using fentanyl 5- -0,3 mg/kg and rocuronium bromide 0,6 mg/kg for tracheal intubation. Patients will be ventilated with volume or pressure controlled ventilation with tidal volume (Vt 6-8 ml/kg ideal body weight), with the intention to maintain end tidal pCO2 (EtCO2) between 4 and 5 kPa. After introduction of GA, in each patient a central venous catheter will be inserted (PreSep Central Venous Oximetry catheter, Edwards, USA) for hemodynamic measurements, as well as MAC (multi-access catheter) for fluid replacement therapy. GA before CPB will be maintained with volatile anesthetics and continuous infusion of opioid analgesic remifentanyl; for maintenance of GA during CPB we will use continuous infusions of remifentanyl and intravenous anesthetic propofol (4-12 mg/kg/h). Standard and extended hemodynamic monitoring will be used during general anesthesia: ECG, invasive arterial blood pressure, central venous pressure, cardiac output, systemic vascular resistance, central venous oxygen saturation, pulse pressure variation, stroke volume variation (EV 1000 platform, Edwards, USA). We will also monitor: hemoglobin oxygen saturation, capnography, cerebral oxygen saturation (NIRS, near infrared spectroscopy), depth of anesthesia (BIS, bispectral index), body temperature, urine output, acid base status with blood gas analysis. We will note type and duration of surgery, duration of CPB, aortic cross-clamping time, blood loss and quantity of crystalloids, colloids and blood components given,

use of inotropic and/or vasoactive drugs, use of insulin. During every procedure we will use transoesophageal echocardiography to assess global cardiac function, regional wall motion abnormalities, valvular function, volume status, as well as for de-aeration before unclamping of the aorta. Type of CPB that we’ll use is standard (type of oxygenator - Inspire (Sorino, Milan, Italy) or Fusion (Medtronic, Minneapolis, USA). Composition of priming solution: ringer, 20% mannitol, heparin. Goals to be achieved during heart surgery for aortic valve replacement in order to achieve adequate cerebral perfusion Hemodynamic management in cardiac patients during surgery is standard and essential. Maintain appropriate blood systemic arterial pressure (systolic 90 to 100 mm Hg, MAP 60 to 65 mmHg) volume load will be guided by central venous pressure, stroke volume variation and transoesophageal echocardiography. Ventilation target Pa CO2 5-5,5 k Pa in arterial blood. Metabolic (insulin/glucose) and coagulation management – routine check-up of arterial blood gas, and transfusion if it is necessary. Detection of embolization during surgical procedure using trans-cranial Doppler We will use TCD (Waki E , Atys Medical, France ) for measuring flow in brain arteries and detect MES in real-time. TCD probe (2kHz) will be set on both sides of the temporal bone in front of ear. Through this acoustic window, we will measure the blood flow in middle cerebral artery on both sides. MES will be determined by TCD during following time-points: before surgery, after sternotomy, between cannulation of, on CPB, during venting of the heart, opening of the aortic clamp and after the completion of CPB . Doppler signals will be included in Doppler device and then analyzed with the program ( TCD - EMB 10.4 Atys Medica, France) on Doppler / personal computer determining the number and type of MES during each period referred above. Intensive care treatment Following surgery, patients will be transferred in cardiac-vascular ICU (CV-ICU) intubated, sedated and mechanically ventilated. They will be awaken and extubated when extubation criteria are fulfilled: awake, cooperative patient, with completely reversed neuro-muscular function, hemoglobin oxygen saturation above 96% with FiO2 0,5 or less, EtCO2 less than 6 kPa, stable hemodynamic and normal core temperature, with retrosternal drainage less than 100 ml/h. Postoperative pain will be treated with continuous intravenous infusion of opioid analgesic morphine and nonsteroidal analgesic metamizol mixture. Measurements We will document demographic characteristics of patients, their preoperative medical status, as well as intraoperative data (type and duration of surgery, duration of CPB, period of ischemia, hemodynamic measurements, usage of inotropic/vasoactive therapy, insulin, fluids, blood and blood components); duration of mechanical ventilation in CV-ICU, duration of intensive therapy, postoperative complications, 30-day mortality. For the purpose of the study, postoperative hemodynamic values will be recorded in the first 5 postoperative days. Determination of biochemical markers. Blood samples will be obtained:

1) Before induction of general anesthesia 2) After completion of CPB 3) After completion of surgery (i.e., on admission in CV-ICU) 4) 24 hours after surgery 5) 48 hours after surgery 6) On 7th postoperative day

The following biochemical markers will be determined:

1. S100B 2. Pro- and anti-inflammatory cytokines: TNF- IL-6, IL-8, IL-10 in plasma ( ELISA) 3. micro particles, exosomes

4. PON1 activity, genotype and phenotype (genotype and phenotype will be determine only from the first patients sample) 5. Lipide status (HDL in LDL holesterol) 6. hs-CRP (high sensitivity CRP), PCT (ECLIA), Differential blood count 7. Classical clinical and microbiological approach will be used to determine possible infection in patient. Postoperative complications: We will record postoperative complications such as: bleeding, hemodynamic instability, impaired respiratory function, worsening of renal, liver and brain functions. Patients will be transferred on ward when following conditions are met: hemoglobin oxygen saturation of 94% or more at FiO2 of 0.5 or less, hemodynamic stability without hemodynamically significant arrhythmias, with diuresis > 0.5 ml/kg/hour, without intravenous inotropic or vasopressor therapy, as well as without delirium or epileptic activity, without signs of infection. Mini mental test will be repeated at 7th days after surgery. Follow-up by telephone will be carried out 30 days after the procedure, when we will focus on late postoperative morbidity and mortality. All personnel included in the study, together with those involved in ICU treatment, will be ‘blinded’ for the assigned treatment throughout whole duration of the study, except the anesthesiologists and perfusionists in operating theatre, who, on the other hand, will not be included in data collection, input and analysis. Expected results

We expect increased occurrence of MES intraoperatively detected by TCD. The difference in the incidence of intraoperative MES should not reach statistical significance when comparing MIS to FS aortic valve surgery. Furthermore, we expect that intraoperative quantity of MES will show positive correlation with postoperative levels of S100B protein as well as with postoperative cognitive decline. We also expect both surgical approaches to impair cerebral-vascular reactivity in the postoperative period, as assessed by VEFR. Summery of expected results for WP1 and WP2 There are two possible major impacts of our proposed project. First, we would like to find the CBP method which would mitigate the SIRS and following excessive CARS. This would results in more hemodynamically stable patients, with less postoperative complications especially less hospital acquired infection, which are one of the major cause of prolonged ICU stay and higher treatment costs and lower quality of life after home discharge and higher mortality. In the future we would like to use PON1 as therapy of immune modulation during/after CBP. Second, MIS AVR has its advantages compared to FS AVR, however the technic could be developed further, especially if we confirm emboli as cause of higher cerebrovascular complications in MIS AVR. Laboratory METHODS for WP1 and WP2: Participating subgroups have proper knowledge and equipment to carry out laboratory analysis and cerebralvascular reactivity testing. PON 1 activity, phenotyping, genotyping (University of Ljubljani, Medical faculty, Institute for biochemistry: prof. Goličnik, prof. Dolžan) Subjects, that will be in the healing process, we will determine the activities and phenotyping of serum PON1 stripped 3-5 ml of arterial blood in tubes without anticoagulant. After the blood clotted, will by centrifugation to remove a blood clot from serum. Thus, serum samples will be prepared within 1 hour freeze at - 20 ° C where they will wait for analysis. PON1 activity in serum samples will be determined spectrophotometrically by measuring the absorbance at the time of the substrate in the presence of phenylacetate (Richter et al, 2008). Phenotyping the PON1 will for each specimen is also determined spectrophotometrically with 2-substrate assay; ie. phenyl acetate and 4-chloromethyl phenylacetate (Richter et al, 2008). In determining genetic polymorphisms PON1 will be to isolate DNA each specimen once stripped of 3-5 ml of arterial blood in a tube with anticoagulant (EDTA or citrate). Blood samples will be frozen at - 20 ° C where they will wait for analysis. After thawing, we collected DNA from blood samples isolated using commercially available whale (Flexigene DNA kit, Qiagen). To determine the genetic polymorphisms PON1 we use a high-performance method based on the polymerase chain reaction (PCR) in real time (TaqMan assay, Applied Biosystems). Lymphocyte Immunophenotyping (University of Ljubljani, Medical faculty, Institute for microbiology and immunology, Laboratory for immunology: prof. Ihan) All monoclonal antibodies (MoAb) were labeled directly either with fluorescein isothiocyanate or phycoerythrin. Non-specific isotype mouse MoAb were used as negative controls. Antibodies against the following cell surface structures were applied: CD3, CD4, CD8, CD19, HLA-DR, NK CD14/CD56, CD4+RA, CD4+RO (Exalpha Biologicals, Boston, MA, USA. The samples (100 µl of blood) were incubated with 10 µl of the appropriate MoAb for 15 to 30 min at room temperature in a dark place then washed twice with cold PBS buffer (Becton Dickinson). Red blood cells were eliminated by adding 2 ml of lysing solution PBS, mixed and centrifuged 5 min at 1600/min. The samples were analyzed on a FACScan flow cytometer (Becton-Dickinson, Heidelberg, Germany) with data stored in list mode files. Expressions of CD64 and CD163 on neutrophils and monocytes (University of Ljubljani, Medical faculty, Institute for microbiology and immunology, Laboratory for immunology: prof. Ihan) Expressions of CD64 and CD163 on neutrophils, monocytes and lymphocytes were measured by quantitative flow cytometry with a FACSCalibur flow cytometer (Becton Dickinson, NY, USA) and FACSCanto flow cytometer (Becton Dickinson, CD, USA) using the Leuko64TM assay (Trillium Diagnostics, LLC, Maine, USA). 50 μL of whole blood, or diluted whole blood to adjust leukocyte concentration to less than 25 x109 /L, was incubated for 15 minutes in the dark at room temperature with a mixture of murine monoclonal antibodies followed by red cell lysis with an ammonium-chloride-based red cell lysis solution (Trillium Lyse). Fluorescence beads were then added and flow cytometer analysis was performed on a minimum of 50,000 leukocytes. Data analysis for fluorescence intensity was performed by CellQuest software (Becton Dickinson, CA, USA). MFI was measured as a linearized value of log scale on lymphocytes (red, negative control, measuring CD64 expression), monocytes (green, positive control, measuring CD64 and CD163 expression), neutrophils (blue, measuring CD64 expression), and beads (aqua blue, measuring FITC and PE expression). Cytokine measurements (University of Ljubljani, Medical faculty, Institute for microbiology and immunology, Laboratory for immunology: prof. Ihan) Cytokine levels in plasma were measured by commercially available ELISA kits. The tumour necrosis factor-alpha (TNF-α) (Milenia Biotec, Germany), Interleukin (IL)-6, IL-4, IL-8, IL-10 and sIL-2 (Thermo Scientific, USA) were measured aaccording to manufacturers instructions.

Analysis of micro particles and exosomes (University of Ljubljani, Medical faculty, Institute for pharmacology: prof. Lipnik-Štangelj, prof. Kržan, dr. Črne and Institute for patophysiology: prof. Marš and dr. Miš) For the analysis of exosomes 3-5 ml of blood will be drown into vacutainersTM with trisodium salt of citrate. Plasma will be centrifuged at low speed and part of the supernatant will be stored at 4 °C for dynamic light scattering analysis (DLS). The DLS analyze fluctuation / oscillation in the intensity of scattered light caused by random Brownian motion of particles

can be calculated, which is a parameter determining the shape of the particles. This parameter confirmed that the measured particles are exosomes, characterized by the weight that is concentrated on the surface (=hollow sphere). From the remaining supernatant, which will not be used for DLS, exosomes will be purify and concentrate by differential ultracentrifugation. Pellet of exosomes resuspended in PBS will be freeze in the vapor phase of nitrogen and stored at -80°C. Exosomes characterization will be performed using flow cytometry. Exosome will first bind to the latex beds and then will be incubated with monoclonal antibodies against specific CD antigen (CD9, CD63, CD81) labeled with fluorochromes. Flow cytometry data will be acquired on Cell Lab Quanta SC-MPL (Beckmann Coulter), since Coulter-principle of electronic volume for the determination of the absolute particle size is more precise than the forward-scatter method which is used alternatively in flow cytometry. Content of exosomes will be analysed by Western-blot and RT PCR methods.

ORGANISATION OF PROJECT The proposed project will be supervised by the Steering Committee and coordinated by Coordinating Group. The Coordinating group will report regularly, every 6 months, on the progress of the study to Steering Committee and annually to the Commission for Medical Ethics of Republic Slovenia (KME RS). In cases of emergency and complication (i.e. severe health complications related to study protocol or even death of a patient) Leaders of WPs will temporary stop the study and within 48 hours report to Coordinating Group and Steering Committee, which will then take appropriate action (i.e. . an extreme consequence is the termination of the study) and notify KME RS and Slovenian Research Agency (ARRS). Steering Committee: prof. Matej Podbregar, Head of Steering Committee, University medical center (UMC) Ljubljana, Slovenia, Clinical department for anesthesiology and surgical intensive care prof. Tomislav Klokočovnik, UMC Ljubljana, Head of Clinical department for cardiovascular surgery prof. Metod Lipnik Štangelj, University of Ljubljana, Medical faculty (ULJ, MF), Institute for pharmacology as.prof. Maja Šoštarič, UMC Ljubljana, Clinical department for anesthesiology and surgical intensive care, Head of Unit for cardiovascular anesthesia Coordinating Group: as.prof. Maja Šoštarič, UMC Ljubljana, Head of Coordinating Group, Clinical department for anesthesiology and surgical intensive care, Head of Unit for cardiovascular anesthesia Gordana Taleska MD MSc¸ UMC Ljubljana, Leader of WP1 Marija Božinovska, MD, UMC Ljubljana, Leader of WP2 as. Andrej Fabjan MD PhD, ULJ MF, Institute for physiology prof. Marko Goličnik, ULJ MF, Institute for biochemistry Prof. Alojz Ihan, ULJ MF, Institute for microbiology and immunology, Head of department for immunology prof. Milan Skitek, UMC Ljubljana, Head of Central Laboratory prof. Tomaž Marš, ULJ MF, Institute for patophysiology prof. Metoda Lipnik Štangelj, ULJ MF, Institute for pharmacology Research subgroup: 1. Cerebral vascular reactivity and transcranial Doppler (ULJ MF, Institute of Physiology: as. Fabjan MD PhD) 2. PON1 activity, phenotyping and genotyping (ULJ MF, Institute of Biochemistry: prof. Goličnik, prof. Dolžan ) 3. Inflammatory response and cellular immunity (ULJ, MF, Institute of Microbiology and Immunology: prof. Ihan) 4. Exosomes and micro particles (ULJ MF, Institute of Pharmacology: prof. Kržan, prof. Štangelj Lipnik and Črne PhD; ULJ MF, Institute for patophysiology: prof. Marš, as. Miš PhD) 5. Other laboratory analysis (UMC Ljubljana, Central laboratory: prof. Skitek, Jerina PhD) Schedule in months (M): 0-2 M: two pilot inclusion of patients in each WP (WP and WP2) for checking/verifying entire logistics routes, measurements and blood tests at 2M: meeting of the Coordination Group which reports to the Steering Committee, and the decision on the beginning of the project 2-12 M: recruiting patients in WP1 and WP2, laboratory analysis at 6M: interim analysis (WP leaders and Coordinating Group report to Steering Committee) at 12 M: interim analysis, meeting and reporting (Coordinating Group reports to the Steering Committee, ARRS and KME RS) 12-24 M: recruiting of patients in WP1 and WP2, laboratory analysis At 18 M: interim analysis (WP leaders and Coordinating Group reports to Steering Committee) At 24 M: interim analysis, meeting and reporting (Coordinating Group reports to the Steering Committee, ARRS and KME RS) 24-32 M recruiting patients in WP1 and WP2, laboratory analysis 32-35 M: final analysis of the data; meeting and reporting, international publication (WP leaders and Coordinating Group) at 36 M: final meeting and report (Coordinating Group reports to the Steering Committee, ARRS and KME RS)

Current project status:

2-12 M: recruiting patients in WP1 and WP2, laboratory analysis at 6M: interim analysis

PUBLICATIONS:

No publications.