ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2020

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1651

Remote Ischemic Preconditioningand its Effects on the RespiratorySystem

ASTRID BERGMANN

ISSN 1651-6206ISBN 978-91-513-0907-1urn:nbn:se:uu:diva-406948

Dissertation presented at Uppsala University to be publicly examined in Enghoffsalen,Akademiska Sjukhuset, Uppsala, Tuesday, 19 May 2020 at 13:00 for the degree of Doctorof Philosophy (Faculty of Medicine). The examination will be conducted in English. Facultyexaminer: Professor emeritus Ola Winsö (Department of Surgical and Perioperative Sciences,Umea University).

AbstractBergmann, A. 2020. Remote Ischemic Preconditioning and its Effects on the RespiratorySystem. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty ofMedicine 1651. 47 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0907-1.

Mechanical ventilation in itself can lead to pulmonary damage, and one-lung ventilation (OLV),necessary for thoracic surgery, accentuates this injury. Remote ischemic preconditioning (RIP)is a potential tool to reduce lung injury after mechanical ventilation, including OLV. However,current data on pulmonary RIP-effects are contradictory. Therefore, the overall purpose ofthis Ph.D. project was to assess the effects of RIP on the respiratory system. In Study I, inhealthy spontaneously breathing volunteers, oxygenation was impaired early after RIP, whichwas possibly induced by transient ventilation-perfusion inequality. Studies II, III, and IV wereperformed in a porcine OLV model. In Study II, we found that RIP possibly enhances alveolarinjury, but attenuates the immune response. In Study III, we confirmed that an immune responseto RIP takes place, which shows a different time pattern in each cytokine, depending on the siteof measurement as well. In Study IV, we studied the porcine model for eight hours and foundthat RIP improved oxygenation after two hours of OLV and impeded the decline of exhalednitric oxide (NO) during and after OLV. These findings indicate that RIP mitigates hypoxicpulmonary vasoconstriction (HPV).

In summary, RIP has a complex effect on the respiratory system, which partly explains theprevious contradictory findings.

Keywords: one-lung ventilation, ventilation-induced lung injury, alveolar immune response,remote ischemic preconditioning, exhaled nitric oxide, animal model

Astrid Bergmann, Department of Surgical Sciences, Anaesthesiology and Intensive Care,Akademiska sjukhuset, Uppsala University, SE-75185 Uppsala, Sweden.

© Astrid Bergmann 2020

ISSN 1651-6206ISBN 978-91-513-0907-1urn:nbn:se:uu:diva-406948 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-406948)

”All sorts of things can happen when you’re open to new ideas and

playing around with things.”

Stephanie Kwolek

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Bergmann, A., Jovanovska, E., Schilling, T., Hedenstierna, G.,

Föllner, S., Schreiber, J., Hachenberg, T. (2020) Early and late Effects of Remote Ischemic Preconditioning on Spirometry and Gas Exchange in Healthy Volunteers. Respiratory Physiology and Neurobiology, 271: 103287.

II Bergmann, A., Schilling, T., Hedenstierna, G., Ahlgren, K.,

Larsson, A., Kretzschmar, M., Kozian, A., Hachenberg, T. (2019) Pulmonary Effects of Remote Ischemic Preconditioning in a Porcine Model of Ventilation-Induced Lung Injury. Respiratory Physiology and Neurobiology, 259: 111-118.

III Bergmann, A., Breitling, C., Hedenstierna, G., Larsson, A.,

Kretzschmar, M., Kozian, A., Hachenberg, T., Schilling, T. (2018) Data on the Effects of Remote Ischemic Preconditioning in the Lungs after One-Lung Ventilation. Data in Brief, 21: 441-448.

IV Bergmann, A., Schilling, T., Perchiazzi, G., Kretzschmar, M.,

Hedenstierna, G., Hachenberg, T., Larsson, A. (2020) Effect of Remote Ischemic Preconditioning on Exhaled Nitric Oxide Concentration in Piglets during and after One-Lung Ventilation. Respiratory Physiology and Neurobiology, 276: 103426.

Reprints were made with permission from the respective publishers.

Contents

INTRODUCTION .....................................................................................11Remote Ischemic Preconditioning .........................................................11Clinical Effects of RIP ..........................................................................11Lung Injury during Mechanical Ventilation ...........................................12RIP and the Lungs .................................................................................12

AIMS OF THE STUDIES .........................................................................15

MATERIALS AND METHODS ...............................................................16Study Protocols .....................................................................................16

Study I ..............................................................................................16Study II and Study III .......................................................................16Study IV ...........................................................................................17

Clinical Study .......................................................................................17Subjects ............................................................................................17RIP / Sham Procedure .......................................................................18Blood Gas Analysis ..........................................................................18Lung Function Tests .........................................................................18

Animal Experiments .............................................................................18Subjects (Studies II – IV) ..................................................................18Anesthesia (Studies II – IV) ..............................................................19Instrumentation (Studies II – IV).......................................................19Ventilation Management (Studies II – IV) .........................................20OLV (Studies II – IV) .......................................................................20RIP / Sham Procedure (Studies II – IV) .............................................20Measurements (Studies II – IV) ........................................................20Sample Collection and Analysis (Study II and III).............................21BAL (Study II and III) ......................................................................21Staining Procedure and DAD Score Assessment (Study II and III) ...22Exhaled NO (Study IV) ....................................................................22

Statistical Analysis ................................................................................22

RESULTS .................................................................................................24Subject Characteristics in Study I ..........................................................24Effect of RIP on Spirometry and Gas Exchange in Study I....................25Hemodynamic and Respiratory Data (Studies II and III) ........................27DAD (Studies II and III) .......................................................................28

Alveolar Cell Numbers (Studies II and III) ............................................29Cytokine Concentration in Lung Samples, BAL Fluid and Serum (Studies II and III) .................................................................................30Exhaled NO (Study IV) .........................................................................31Oxygenation (Studies II and III) ............................................................32Oxygenation (Study IV) ........................................................................32

DISCUSSION ...........................................................................................33Summary of Main Results .....................................................................33The Effects of RIP on Oxygenation in Healthy Subjects without any Intervention...........................................................................................33The Effects of RIP on Oxygenation in Pigs............................................35The Effects of RIP on Alveolar Damage ................................................35The Effects of RIP on the Immune Response .........................................35The Effects of RIP on BAL Fluid ..........................................................37The Effects of RIP on Exhaled NO ........................................................37Limitations of the Clinical Study ...........................................................39Limitations of the Experimental Studies ................................................39

CONCLUSIONS .......................................................................................40

ACKNOWLEDGEMENTS .......................................................................41

REFERENCES ..........................................................................................42

Abbreviations

ARM alveolar recruitment maneuver BAL broncho-alveolar lavage BMI body-mass index CO carbon monoxide DAD diffuse alveolar damage DAMP damage-associated molecular pattern DLCO diffusing capacity for carbon monoxide DLNO diffusing capacity for nitric oxide Dm membranous component ELISA enzyme-linked immunosorbent assay FIO2 fraction of inspired oxygen FMD flow-mediated dilation Hb hemoglobin HMGB high-mobility group box H&E hematoxylin and eosin H2O2 hydrogen peroxide HPV hypoxic pulmonary vasoconstriction ICA internal carotid artery ID inner diameter IL interleukin IRI ischemia-reperfusion injury KATP-channel potassium-dependent channel for adenosine triphosphate mRNA messenger ribonucleid acid MV mechanical ventilation NF nuclear factor NO nitric oxide NO2

- nitrite ion NO3

- nitrate NOS nitric oxide synthase PAC pulmonary artery catheter pAO2 partial pressure of oxygen in the alveolae PAP pulmonary arterial pressure PASP pulmonary arterial systolic pressure pCO2 partial pressure of carbon dioxide in arterial blood PEEP positive end-expiratory pressure PKC protein kinase C

pO2 partial pressure of oxygen in arterial blood PVR pulmonary vascular resistance Qs/Qt intrapulmonary shunt / venous admixture RAGE receptor for advanced glycation end products RIP remote ischemic preconditioning ROS reactive oxygen species SaO2 arterial oxygen saturation SH-group sulfhydryl group SIRS systemic inflammatory response syndrome TLR toll-like receptor TLV two-lung ventilation TNF tumor necrosis factor Vc capillary volume VT tidal volume

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INTRODUCTION

Remote Ischemic Preconditioning Remote ischemic preconditioning (RIP) is defined as brief episodes of ischemia – usually performed with a blood pressure cuff on one limb – followed by reperfusion after releasing the cuff. This cycle is repeated three or four times, depending on the protocol of the experiment. RIP is expected to protect remote target organs, for example the heart, the brain or the kidneys, from ischemia-reperfusion-injury (1). However, current experimental data and clinical results on RIP effects are contradictory.

It is still unclear how RIP works, but its mechanism is supposed to act in different ways. Inflammatory mediators like TNF-α or interleukins (IL) like IL-1, IL-6 and IL-10 and their receptors might be involved as well as activation of the NF-κB pathways. The activation of neuronal mechanisms is also discussed. The expression of anti-oxidative enzymes and the suppression of reactive oxygen species (ROS) seem to play a key role as well as the transcription and expression of anti-inflammatory mediators (2, 3). These mediators activate intracellular pathways after binding to their receptors, leading to the opening of potassium-dependent adenosine triphosphate (KATP)-channels and preventing permeability-pores from opening, thus protecting the mitochondria and lowering the electric potential of the membrane. Eventually, the cells will activate their self-repair-mechanisms and improve their survival, preventing ischemic injury and thus inflammation and apoptosis (4, 5).

Clinical Effects of RIP There are clinical studies with different outcomes after RIP. Present data are inconsistent regarding improved or even worse outcomes for the reason of different patient populations, methodology and measurement. The prehospital application of RIP has been shown to increase myocardial salvage in patients with acute coronary syndrome (6) but large clinical studies in cardiac surgical patients failed to demonstrate an improved postoperative outcome (7, 8). The rate of acute kidney injury was significantly reduced with RIP (9) in high-risk cardiac surgery patients but RIP exerted no clinical benefits for cerebral and cardiac protection during carotid endarterectomy (10).

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Lung Injury during Mechanical Ventilation Mechanical ventilation in itself can lead to pulmonary damage in sick lungs (11, 12) but also in healthy ones (13, 14). Elevated airway pressures and shear stress that come up after the collapse of alveoli and their re-opening might be responsible for this. Overall, alveolar and interstitial edema, neutrophil infiltration, alveolar overdistension, microhemorrhage and microatelectasis are the main issues in diffuse alveolar damage (DAD) (15). An increase in DAD scores can be observed as well as inflammatory responses assessed by elevated cytokine expressions in serum and lung tissue after MV. Besides, mechanical ventilation increases leucocyte recruitment into the lungs (16-18).

The alveolar damage is even accentuated when one-lung ventilation (OLV) (8), necessary for thoracic surgery, is applied (19-22). During OLV, high minute volumes and elevated airway pressures might result in an inflammatory reaction in the ventilated lung. Therefore, patients undergoing OLV are more likely to face pulmonary complications in the postoperative setting than patients who had experienced normal two-lung-ventilation. A clinical study found a correlation between the duration of OLV and the extent of oxidative lung injury (23).

When TLV is re-established, re-ventilation and re-perfusion result in alveolar damage in the sense of ischemia-reperfusion injury. The combination of OLV and surgery dramatically harms the lung, and the resulting damage can be assessed in terms of the DAD score (24, 25).

Several clinical studies revealed that lung resection surgery is associated with increased markers of oxidative stress and inflammation (26-28). Previous data obtained by bronchoscopic microsampling revealed that IL-1β, IL-6, and IL-8 are significantly increased both in the ventilated and in the non-ventilated lung at the end of lung resection surgery (29). The production and release of superoxide anions and cytokines may be induced by hypoxic pulmonary vasoconstriction (HPV) in the collapsed lung. OLV has been shown to increase concentrations of 8-isoprostane, NO2- and NO3- and H2O2 in exhaled breath condensate (30).

In patients undergoing lung surgery, pulmonary complications represent a major risk for increased postoperative morbidity and mortality (31). Intraoperative OLV may also contribute to lung injury (32).

RIP and the Lungs Several researchers have demonstrated that RIP does affect pulmonary function in a positive way: Xia et al. showed that pulmonary vascular resistance (PVR) was decreased after RIP and hinted that KATP-channels were responsible for pulmonary vasodilation, leading to a reduction in pressure and hence protecting the lung (33). Harkin et al. analyzed PAP, pAO2, pO2, MPO-

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activity and lung weight after RIP (34) and discovered that IL-6 was reduced as well as circulating phagocytes and neutrophil recruitment into the lungs were diminished, indicating a protective effect to the lungs. Cheung et al. presented data that RIP did not compromise lung function and reduced cytokine levels (35, 36).

Waldow and his colleagues confirmed as well that IL-6 levels were lower after RIP and demonstrated that lung ischemia-reperfusion injury was reduced as shown by an uncompromised pulmonary function, gas exchange and non-elevated pulmonary tension (37, 38). All indices of severe inflammatory response syndrome (39) (ROS, granulocytes, etc.) were not modulated. In that study, RIP totally prevented the deterioration of pulmonary function and the incidence of pulmonary hypertension. The lung function was not compromised, showing that RIP had a preserving effect towards the lung.

Li et al. analyzed patients undergoing partial lung resection for lung carcinoma in OLV (40). They provided RIP to some of them and found a significant decrease in lung injury among those patients submitted to RIP. Their primary outcome was oxygenation (pO2/FIO2), secondary outcomes being pulmonary variables, the incidence of postoperative complications, markers of oxidative stress and inflammatory response. RIP performed before OLV and surgery significantly increased pO2/FIO2 at all time points where it was determined compared to the control group, hinting at improved oxygenation.

It was suggested by the authors (40) that the ischemia-reperfusion injury after OLV resulted from atelectasis and re-expansion leading to elevated biochemical markers and decreased lung-function due to an inflammatory response. These markers were supposed to be released after the collapsed non-ventilated lung was re-opened, because this side was not only non-ventilated but also non-perfused due to HPV. Free oxygen radicals were discharged into the circulation, aggravating the inflammatory reaction.

In 2015, a long expected multicenter trial on RIP ended and the results were published (7). Against all expectations, no effect could be shown concerning the incidence of myocardial infarction, new stroke or renal failure in patients after cardiac surgery up to hospital discharge, which were the primary endpoints. Besides, secondary endpoints were determined as the incidence of the primary ones after 30 and 90 days after surgery. No significant differences between groups were established. Minor parameters that were published included pulmonary variables like oxygenation, length of mechanical ventilation (41), or need for re-intubation, which did not show significant differences between the RIP group and the control group neither. The multicenter trial showed no effect on hospital dismissal, which was roughly two weeks after surgery, and 30 and 90 days after surgery.

Different approaches have been applied to attenuate the pulmonary injury. Mechanical ventilation with reduced tidal volumes (VT) (42) and positive end-expiratory pressure (PEEP) (43, 44) did not completely inhibit pro-

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inflammatory responses in rats and rabbits (45). However, reduced VT and decreased peak airway pressures had a significant protective effect on the alveolar inflammatory response after OLV in patients (46). Therefore, other methods are needed to prevent postoperative pulmonary complications.

The effects of RIP on the lungs have only been assessed in very few studies. A major problem in clinical studies is that tissue samples representing different regions of the lungs cannot be obtained in thoracic surgical patients. For this reason, potential protective effects of RIP on the ventilated, non-operated lung and on the non-ventilated, collapsed lung in the setting of thoracic surgery as well as the effects of RIP on healthy lungs have not yet been studied in general.

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AIMS OF THE STUDIES

The objectives of the present clinical and experimental studies are: 1. To analyze the effects of RIP on pulmonary function, on gas

exchange, and on inflammatory responses in healthy, spontaneously breathing volunteers.

2. To assess the effects of RIP on respiratory function and pulmonary tissue integrity as well as on alveolar injury in a porcine model.

3. To assess the effects of RIP on the immune response in the context of mechanical ventilation including OLV in different tissues.

4. To evaluate whether the effects of RIP remain throughout a prolonged period of mechanical ventilation.

5. To analyze the role of RIP on exhaled nitric oxide (NO) before and after OLV in a porcine model.

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MATERIALS AND METHODS

Study Protocols Study I As there are not data on the effects of RIP on pulmonary function in healthy people who do not undergo any intervention, this prospective study was undertaken to assess the effects of RIP after one and 24 hours on gas exchange and lung function. In the control state (T1) hemodynamic data (heart rate and blood pressure) were obtained. Capillary blood from an earlobe was taken. Then pulmonary function testing, including spirometry, body plethysmography and diffusion measurement were performed. The measurements were repeated 60 minutes (T2) and 24 hours (T3) after RIP. Between T1 and T2, all study subjects remained in a semi-supine position with the upper body slightly raised and legs stretched. They were awake and did not have food or drinks during that time. Between T2 and T3, the study subjects followed their individual daily routine, but abstained from vigorous exercise, caffeine and alcohol and were asked to consume a light meal two hours before coming to the lung function test laboratory.

Study II and Study III The objective of this prospective, randomized, controlled experimental study was to assess the effects of RIP on respiratory function and pulmonary tissue integrity as well as on alveolar injury in a porcine model of injurious ventilation. The Animal Ethics Committee of Uppsala (Sweden) approved the experimental protocol.

Fourteen piglets were used in the study. The pigs were randomized to either receive RIP before OLV or not, and induction to general anesthesia was performed in the same fashion in all animals. Ventilator settings were the same in both the RIP and the control group during OLV as well as during TLV.

After the end of the protocol the entire lungs were excised immediately to obtain histological samples.

Broncho-alveolar lavage (47) fluid and serum samples were collected according to the timepoints illustrated in figure 1. BAL fluid was used for cell counting, whereas in the serum samples cytokines were determined using commercially available quantitative sandwich enzyme-linked immunosorbent

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assays (ELISA). The histological samples of the lungs were used to assess the DAD score.

Two manuscripts were published that include data from this experiment.

Fig. 1: Timeline for Studies II and III.

Study IV Few experimental data are available on exhaled markers after RIP and on the prolonged effects of RIP on the lung. Therefore, in the fourth study a protocol was applied focusing on respiratory parameters and on exhaled NO before, during and after OLV in a porcine model. The objective of this prospective, randomized, controlled study was to assess cardiopulmonary variables, gas exchange and exhaled NO up to eight hours after OLV in juvenile piglets subjected to RIP or a sham protocol before OLV. The timeline of the protocol is illustrated in figure 2.

Fig. 2: Timeline for Study IV.

Clinical StudySubjectsThirteen healthy non-smoking study subjects were included in the RIP group, whereas twelve matching controls were allocated to the control group. The Ethics Committee of the Otto-von-Guericke-University Magdeburg (Germany) approved the experimental protocol and all study subjects had given written informed consent to participate in the study.

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Exclusion criteria were the following: obesity as assessed by body-mass index (BMI) more than 30; arterial occlusion disease; regular intake of beta-blocking agents, calcium antagonists, phosphodiesterase inhibitors, xanthines; nocturnal hypoxemia / hypercapnia, i.e. sleep apnoea. Individuals with persistent tobacco abuse and with evidence of pulmonary or systemic infections (clinically defined) were also excluded.

RIP / Sham Procedure RIP was performed by inflating a blood pressure cuff placed around the femoral part of one lower limb to a pressure 20mmHg above the systolic blood pressure as assessed by Doppler ultrasound. Complete cessation of arterial perfusion was verified by pulse oximetry of the great toe and by Doppler ultrasound. After five minutes the blood pressure cuff was released for five minutes rest. The procedure was repeated three times using the same leg resulting in 40 minutes ischemia and reperfusion. The control group underwent the same protocol without inflation of the blood pressure cuff.

Blood Gas Analysis Capillary blood was collected from a hyperemised earlobe. Blood gas analysis for determination of oxygen tension (pO2) and carbon dioxide tension (pCO2) was performed (Abl90flex Fa. Radiometer, Germany). Capillary pO2 and pCO2 have been demonstrated to reflect arterial blood gases with sufficient precision and are regularly used for lung function tests in volunteers and patients (48, 49).

Lung Function Tests Pulmonary function tests including spirometry, flow-volume-loop and body plethysmography were performed using the MasterScreenPro®, Fa. Viasys, Germany. Pulmonary diffusion capacity was measured with the single breath technique using carbon monoxide (CO) and NO as test gases (Fa. Viasys MS PFT Pro®). The components of pulmonary diffusion capacity, capillary volume (Vc) and membranous component (Dm) (7) were calculated based on these measurements.

Animal Experiments Subjects (Studies II – IV) The experiments were conducted in accordance with the National Institutes of Health guidelines for ethical animal treatment. The Animal Ethics Committee

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of Uppsala (Sweden) approved the experimental protocol. The studies were carried out as prospective, randomized, controlled animal experiments.

A total of twenty-six 2-3-month-old Norwegian Landrace-Yorkshire crossbred piglets (26±2kg) obtained from a local breeder were used. The animals fasted overnight and had free access to water. All pigs underwent the same study protocol (anesthesia, instrumentation, measurement algorithms). The animals were randomly assigned to either a RIP or a control group (EXCEL®, Microsoft Corp., USA) in the Studies II, III and IV.

Anesthesia (Studies II – IV) General anesthesia was induced by an i.m. injection of xylazine (2.2mg·kg-1, Rompun®; Bayer, Leverkusen, Germany) and tiletamine / zolazepam (6mg·kg-1, Zoletil®; Virbac, Carros, France). After testing for the absence of corneal reflexes and hind limb flexion reflex response, ear veins were cannulated to administrate fentanyl (up to 4μg·kg-1). Animals were placed supine and tracheostomy with an ID 8.5 mm endotracheal tube (Mallinckrodt, Athlone, Ireland) and cannulation of major vessels were performed.

The animals were kept in supine position throughout the experiment. Anesthesia was maintained by continuous i.v. infusion of fentanyl (0.04mg·kg-1·h-1, Leptanal®; Janssen-Cilag AB, Sweden), midazolam (0.12mg·kg-1·h-1, midazolam Actavis, Actavis Group, Hafnersfjordur, Iceland) and propofol (Diprivan®; Astra, Södertälje, Sweden), and muscle relaxation was achieved with a continuous intravenous infusion of 2.5mg·kg-1·h-1 rocuronium bromide (Esmeron®, N.V. Organon, Oss, Netherlands).

All pigs were killed through an intravenous bolus injection of potassium chloride (150meq) at the end of the study.

Instrumentation (Studies II – IV) A flow-directed pulmonary artery catheter (PAC) (7.0 French, Swan-Ganz thermodilution catheter, Baxter, Irvine, CA, USA) and a central venous catheter (4.0 French, Becton-Dickinson Critical Care Systems, Singapore) were inserted via the right external jugular vein. The PAC was used for cardiac output measurements and mixed venous blood sampling. The PAC was repositioned before each experimental step to ensure that the tip was always located in regions with high pulmonary blood flow.

In addition, the pigs received a right carotid arterial catheter for continuous arterial blood pressure measurements and for blood sampling (20G; Becton-Dickinson Critical Care Systems). Finally, a suprapubic urinary catheter (Sympakath®; Ruesch AG, St. Gallen, Switzerland) was placed to monitor urine output.

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Body temperature was monitored and kept constant by thermoconvection. Pigs were allowed to stabilize for 30 minutes after instrumentation.

Ventilation Management (Studies II – IV) Pressure controlled ventilation was applied throughout the procedure with FIO2 of 0.4 and PEEP of 5cmH2O, provided by a KION® anesthesia ventilator (Maquet Critical Care, Solna, Sweden). VT 10ml·kg-1, and respiratory frequencies were adjusted to achieve a pCO2 of 40mmHg. Fresh gas flow, airway pressures and gas concentrations were measured at the proximal end of the endotracheal tube with a standard monitor for ventilation and hemodynamic measurements (SC 9000 XL, Siemens, Erlangen, Germany).

OLV (Studies II – IV) A left-sided endobronchial blocker (9.0 French Arndt Endobronchial Blocker Set, COOK®, Bjaeverskov, Denmark) was inserted and secured in the left main bronchus. The blocker was inflated under fiber-optic control. The ventilation of the left lung was thus discontinued for 120 minutes (OLV period). Ventilation settings remained unchanged and the VT of 10ml·kg-1 was delivered exclusively to the dependent lung during OLV. In Study IV, FIO2 was set to 1.0 during OLV, whereas it remained at 0.4 in Studies II and III. After two hours of OLV, the lungs were re-inflated by a constant airway pressure of 40cmH2O, applied to the whole lung for 10 seconds (alveolar recruitment maneuver, ARM). Thereafter, TLV was resumed, with the ventilator settings remaining the same as described before.

RIP / Sham Procedure (Studies II – IV) Animals that had previously been randomized to the RIP group had a blood pressure cuff at the left hind limb inflated up to 200mmHg for five minutes followed by five minutes of reperfusion after deflating the cuff, this being repeated four times. The animals randomized to the control group experienced a sham procedure without inflating the blood pressure cuff.

Measurements (Studies II – IV) Ventilation and hemodynamic variables were recorded, and arterial and mixed venous blood samples were taken during baseline TLV, after the RIP-procedure before OLV, after 60 minutes and after 120 minutes of OLV, and 60 minutes after OLV in the Studies II and III (figure 1). The same variables and samples were collected during baseline TLV, immediately after the RIP-procedure and before OLV, after 60 minutes and 120 minutes of OLV, after the first and second hour of TLV and finally two-hourly until the end of the

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protocol in Study IV (figure 2). The samples were taken for blood gas analysis (ABL800, Radiometer, Copenhagen, Denmark), determination of oxygen saturation and hemoglobin (Hb) concentration (OSM 3, Radiometer, Copenhagen, Denmark). Blood gas measurement was corrected for pig blood. Cardiac output was assessed with the PAC. Venous admixture (QS/Qt) was calculated according to the standard formula.

Sample Collection and Analysis (Study II and III) The entire lungs were excised via median sternotomy immediately after the pigs were killed. Blocks of lung tissue of approximately 0.125cm3 were harvested from both lungs’ largest diameter at three locations: from subpleural, intermediate and parahilar regions. Three samples from each location were obtained, immediately frozen in liquid nitrogen and stored at -80°C. Tissue pieces were homogenized in lysis buffer (15mM Tris, pH 7.4, 150mM NaCl, 1mM CaCl2, 1mM MgCl2, 0.5% Triton X-100), with addition of protease inhibitor (Thermo Scientific, Waltham, MA) using an Ultra-Turrax T8 homogenizer. The homogenates were centrifuged at 300RPM at 4°C for 10minutes.

Total protein content of tissue homogenates was measured by Coomassie Plus Assay (Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions. Cytokine concentrations were assessed in serum samples and in tissue homogenates using Quantikine ELISA (R&D systems, Minneapolis, MN), for porcine TNF-α, IL-8, IL-10 and IL-1β/IL-1F2 according to the manufacturer’s instructions.

BAL (Study II and III) Fiber-optic bronchoscopy and BAL (bronchoscope EF-B 14/L, Xion Ltd., Garching, Germany) were performed at defined time points (figure 1). Before and after each manipulation, both lungs were recruited by alveolar recruitment maneuver.

BAL was based on a standardized procedure: The tip of the bronchoscope was brought into wedge position in a segmental bronchus of the left and right lower lung lobes, respectively. For each BAL, a different segmental bronchus was randomly used. For BAL, 40ml of sterile, isotonic saline solution (Fresenius Kabi AB; Halden, Norway) was sequentially instilled (10ml portions) and gently suctioned; ~50% of the fluid was recovered. BAL fluid was collected in sterile tubes and immediately placed on crushed ice.

The lavage fluid was filtered through sterile gauze filters and collected on ice in siliconized containers. 50µl of BAL fluid was used for staining and cell counting (Neubauer cell counting chamber). The remaining fluids were centrifuged at 5000RPM at 4°C for 10min.

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Cell pellets were suspended in ice-cold phosphate buffer containing 0.01% sodium azide and 2% bovine serum and were shock frozen in liquid nitrogen. The cells were stored at -80°C.

Staining Procedure and DAD Score Assessment (Study II and III) Paraffin-embedded lung tissue samples were sectioned (2-3µm slices) and stained with hematoxylin and eosin (H&E) for light-microscopic analysis. The sections were randomly selected by an assisting technician blinded to the experimental protocol. The slides were evaluated by a blinded pathologist. The extent of histomorphological changes was scored by the DAD score.

The alveolar damage was assessed by light microscopy (Model CHK; Olympus, Taiwan). The alveolar injury was characterized by the following features: Alveolar edema, interstitial edema, microhemorrhage, neutrophil infiltration, microatelectasis and alveolar overdistension. Four isolated non-overlapping fields of view of the different samples were analyzed separately. The values of all sectors per lung (n=12) were averaged.

The severity of the DAD features was characterized as follows: 0=normal appearance, 1=slight effect, 2=intermediate effect and 3=severe effect. The extent of alveolar damage in each sector was described as follows: 0=no damage, 1=up to 25%, 2=25–50%, 3=50–75%, 4=75% to almost complete and 5=complete. Each property was given by its severity multiplied by the extent. The DAD score was calculated by summarizing the products of severity and extent of each DAD quality.

Exhaled NO (Study IV) The exhalation of NO was measured at the timepoints mentioned above (figure 2) with a Nitric Oxide Analyzer (Sievers NOA280, GE, Boulder, CO, USA) using the ozone-chemiluminescence technology. The NOAnalysis REB program was used for flow meter calibration, data collection and processing.

Statistical Analysis Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS, v. 24, IBM Corporation, Armonk, New York, USA) and SigmaPlot® v. 13 (Systat Software Inc., San Jose, CA, USA).

Study I: The estimation of sample size was based on previous studies, which used a similar experimental setup. Power calculation using a two-sided design at a significance level of 5% (α=0.05) and a power of 80% (β=0.20) revealed that at least 10 study subjects were needed to detect a difference of

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more than 10% in gas exchange and spirometry variables between study subjects before and after RIP.

Studies II and III: The estimation of sample size was based on previous experimental studies (15, 22), which used an analogous experimental setup. Power calculation using a two-sided design at a significance level of 5% (α=0.05) and a power of 80% (β=0.20) revealed that at least five animals per group were needed to detect a difference of more than 25% in alveolar cytokine concentrations in piglets prior to and after OLV.

Study IV: The estimation of sample size was based on previous experimental studies (15, 22), which used an analogous experimental setup. Power calculation using a two-sided design at a significance level of 5% (α=0.05) and a power of 80% (β=0.20) revealed that at least five animals per group were needed to detect a difference of more than 25% in exhaled NO in piglets prior to, during and after OLV.

All Studies: All data were tested for normal distribution with the Shapiro-Wilk test and are presented as means and standard deviations in the case of normal distribution (cardiopulmonary and ventilation variables). In case of non-normal distribution, results are displayed as box plots (median and interquartile range (IQR, P25-P75). The analysis of normally distributed data was performed by repeated measures of one-way analysis of variance (28) with post-hoc Bonferroni correction.

Study I: Non-normally distributed data were logarithmically transformed to achieve homogenous variances of data sets. Subsequent between-group comparisons were performed by two-way ANOVA using the independent variables “group” and “time”. Post-hoc multiple comparisons were done by the Bonferroni procedure. Study II and III: A repeated measures general linear model (type III sums of squares) assessed the sequential changes of cytokine concentrations in each group. Subsequent between-group comparisons were performed by two-way ANOVA using the independent variables “group” and “time”. Post-hoc multiple comparisons were performed by the Bonferroni procedure. The concentrations of TNF-α and IL-8 remained heteroscedastic even after logarithmic transformation. Therefore, non-parametric Friedman´s test and subsequent Kruskal-Wallis H-Test with adjustment of α-levels for repeated measurements were used to analyze these data sets. In some cases, mediator concentrations were below the detection limits of the immunoassays. These data were included into the statistical analysis with a value of 0.1.

All Studies: No data were lost during the experiment or were missed in the statistical analysis. The differences were considered to be statistically significant for all procedures if p<0.05.

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RESULTS

Subject Characteristics in Study I Biometrical data were similar in all subjects and are illustrated in table 1.

Tab. 1: Subject Characteristics.

Hemodynamic data were normal before and after RIP and after the sham procedure in both groups without significant alterations among and between groups and are illustrated in table 2. Tab. 2: Hemodynamic Data.

BP blood pressure; HR heart rate

j

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Effect of RIP on Spirometry and Gas Exchange in Study I Spirometry showed no differences between T1, T2 and T3 and between groups. All parameters of pulmonary diffusion including the components Vc and Dm did not show any differences between the time points in both groups and are illustrated in table 3.

Tab. 3: Lung Function.

VC vital capacity; FEV 1 forced expiratory volume in one second; FVC forced vital capacity; MEF maximal expiratory flow of the FVC; TLC total lung capacity; RV residual volume; DLCO diffusion capacity for carbon monoxide; Dm membrane factor; Vc capillary volume

g

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60 minutes after RIP, capillary blood pO2 had decreased from a baseline mean of 93.5mmHg (T1) to a mean of 84.5mmHg (T2) in the RIP group (p<0.05), without alterations of capillary pCO2. Twenty-four hours after RIP (T3), capillary blood pO2 had increased to a mean of 91.1mmHg and was significantly higher than at T2 and no longer different from initial baseline (figure 3). The pattern of an early decrease with return to baseline within 24 hours was similar in all 13 subjects in the RIP group.

Fig. 3: Box Plot of pO2 (left) and pCO2 (right) in the RIP group. * indicates the difference between T2 and T1 (p ≤ 0.05). # indicates the difference between T3 and T2 (p ≤ 0.05). No alterations of pO2 and pCO2 were observed in the control group (figure 4).

Fig. 4: Box Plot of pO2 (left) and pCO2 (right) in the control group. There were no gender-specific differences in pO2 at all time points in both groups. Even though there was a trend towards a higher pO2 among the female study-subjects and the decline from T1 to T2 in the RIP group was more accentuated in females, this difference between the genders did not reach statistical significance. pCO2 among the female study-subjects was lower.

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Hemodynamic and Respiratory Data (Studies II and III) The mean values of hemodynamic and respiratory data are shown in the tables 4 and 5. The variables mean pulmonary artery pressure (MPAP), pulmonary artery occlusion pressure (PAOP) and intrapulmonary venous admixture (QS/Qt) remained constant during TLV but increased during and after OLV, without any differences between the RIP and the control group. MAP was slightly but significantly increased after RIP when compared to controls. In both groups, MAP was elevated when compared to baseline.

Peak and mean airway pressures increased in both groups during OLV. Hemodynamic and respiratory variables returned to the initial values when back to conventional TLV at the end of OLV.

Tab. 4: Hemodynamic Data. * indicates differences between the control and the RIP group (p<0.05). # indicates differences as compared to baseline values (p<0.05).

MAP mean arterial pressure; MPAP mean pulmonary artery pressure; CVP central venous pressure; PAOP pulmonary occlusion pressure; CO cardiac output; HR heart rate; SvO2 mixed venous oxygen saturation

Tab. 5: Respiratory Data. * indicates differences between the control and the RIP group (p<0.05). # indicates differences as compared to baseline values (p<0.05).

paO2 arterial partial pressure of oxygen; paCO2 arterial partial pressure of carbon dioxide; PAW airway pressure; PEEP positive end-expiratory pressure; MV minute ventilation; VT tidal volume

g p

ggg p (p ) p (p )

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DAD (Studies II and III) MV including a period of OLV for 120 minutes resulted in a significant increase of global DAD score values in all pigs without any differences between the experimental groups. The median DAD score was 29.8 (range 28.3 - 32.1) in control piglets and 30.4 (range 26.5 - 32.6) in animals with RIP intervention (figure 5).

Fig. 5: Box Plot of DAD scores in the control and in the RIP group, both for the non-ventilated and the ventilated lung. However, in the ventilated lungs of RIP pigs, the items “alveolar edema” and “microhemorrhage” were more pronounced than in control animals (figure 6). The mean values for alveolar edema were 5.4 (range 4.5 - 5.7) in controls vs. 6.0 (range 5.5 - 6.5) in RIP pigs (p < 0.05), and for microhemorrhage (controls vs. RIP) 3.5 (range 2.4 - 4.4) vs. 4.8 (range 4.5 - 5.4; p<0.05).

Fig. 6: Box Plot of two aspects of the DAD score. * indicates differences between the control and the RIP group (p<0.05).

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Alveolar Cell Numbers (Studies II and III) Mechanical ventilation itself increased the number of cells in the BAL fluids, which represents the recruitment of leukocytes in the lung periphery (distal airways and alveoli). However, cell recruitment was significantly higher in the lungs of control animals in comparison with the RIP group, at the end of the study period. In addition, these differences were more pronounced in the right, ventilated lung (figure 7).

Fig. 7: Box Plot of BAL cells in the non-ventilated lung (left) and in the ventilated lung (right). * indicates differences between the control and the RIP group (p<0.05). # indicates differences as compared to baseline values (p<0.05).

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Cytokine Concentration in Lung Samples, BAL Fluid and Serum (Studies II and III) In the lysates of lung tissue samples, the concentration of TNF-α was more increased in controls as compared to RIP pigs, and the ventilated part of the lungs was more affected. This is illustrated in figure 8.

Fig. 8: Box Plot of TNF-α levels. * indicates differences between the non-ventilated and the ventilated lung in the control group (p<0.05). # indicates differences between the control and the RIP group in the ventilated lung (p<0.05).

The total protein content was lower in lung tissue of the RIP group [controls vs. RIP: 11.6 mg/ml (IQR 8.5 - 19.9) vs. 7.2mg/ml (IQR 6.3 - 7.7), p<0.05]. This was accompanied with highest IL-8 concentrations in the non-ventilated lungs of RIP pigs, illustrated in figure 9.

Fig. 9: Box Plot of TNF-α levels. * indicates differences between the non-ventilated and the ventilated lung in the both groups (p<0.05).

Accordingly, serum IL-8 and IL-1β but not TNF-α increased after OLV; however, there was no statistical difference between controls and RIP pigs. The development of cytokines in serum and in BAL fluid confirm that an immunological response to RIP takes place which shows a different time

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pattern in each cytokine, depending on the site of measurement as well (either serum or lungs).

Exhaled NO (Study IV) The levels of exhaled NO are shown in figure 10 for the control group and for the RIP group. During OLV, exhaled NO declined in the control group when compared to baseline levels and remained at this low level until the end of the experiment. In the RIP group, the levels of exhaled NO did not show any significant alterations at any timepoint when compared to baseline.

Fig. 10: Box Plot of exhaled NO in the control (left) and in the RIP group (right). * indicates the difference within the control group as compared to baseline values (p ≤ 0.05).

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Oxygenation (Studies II and III)

All animals had a similar decrease in oxygenation as compared to the pre-OLV data, and both groups showed a trend back to baseline pO2 values during the succeeding TLV. Nevertheless, oxygenation as assessed by pO2 was higher after RIP intervention, and it remained elevated in those pigs that underwent the RIP procedure. Oxygenation was significantly increased in the RIP group at the end of the protocol as illustrated in figure 11.

Fig. 11: Box Plot of pO2. * indicates differences between the control and the RIP group (p<0.05).

Oxygenation (Study IV) pO2 is shown in figure 12. It increased during OLV in both groups. After two hours of OLV, pO2 was higher in the RIP group.

Fig. 12: Box Plot of pO2 in the control and the RIP group. * indicates the difference between the control and the RIP group (p ≤ 0.05).

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DISCUSSION

Summary of Main Results Study I: 60 minutes after RIP capillary pO2 decreased significantly and returned to baseline level after 24 hours. This did not occur in the control group. Capillary pCO2, variables of lung function tests and pulmonary capillary blood volume remained unchanged throughout the experiment in both groups. Biometrical data and hemodynamic parameters were similar between the groups and within the subjects.

Studies II and III: Hemodynamic and respiratory data were similar in both groups. RIP improved oxygenation after OLV, indicated by higher pO2 levels in the RIP group. DAD scores were high without any differences between controls and RIP. On the other hand, alveolar edema and microhemorrhage were significantly increased after RIP. RIP attenuated alveolar recruitment of leukocytes after OLV and TNF-α concentrations in the ventilated lung. The development of cytokines in serum and BAL fluid confirm that an immunological response to RIP took place, which showed a different time pattern in each cytokine, depending on the site of measurement as well (either serum or lungs).

Study IV: Hemodynamic and respiratory data were similar in both groups. Arterial pO2 was higher in the RIP group after two hours of OLV. In the control group, exhaled NO decreased during OLV and remained at low levels for the rest of the protocol. In the RIP group, exhaled NO decreased as well during OLV and was lower when compared to controls but returned to baseline levels when TLV was re-established.

The Effects of RIP on Oxygenation in Healthy Subjects without any Intervention This prospective, randomized study was designed to assess early and late effects of RIP on spirometry and gas exchange in healthy volunteers. So far, no spirometry variables have been published on healthy volunteers prior to and after RIP. Rieger et al. found no alterations of expired minute volume and end-tidal pCO2 in young adults subjected to RIP or a sham protocol during normoxia or hypoxia at sea level (50). These data support the conclusion that static and dynamic variables of lung function are not affected by RIP.

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Measurement of lung diffusing capacity for CO (DLCO) and for NO (DLNO) using a rebreathing technique takes advantage of the diffusion-limited characteristics of CO and NO. In a computed model, Hughes and Dingh-Xuan found a curvilinear relationship between DmCO/Vc and DLNO/DLCO, which was independent of the absolute values of DLNO and DLCO (51). Our results for Vc in both groups are in accordance with their data and reference values for young adults (52). RIP had no early or late effect on DLNO/DLCO ratio or VC despite the fact that different signal triggers have been attributed to alterations of organ blood flow and capillary recruitment. In contrast, oxygenation was slightly impaired early after RIP, but not in the control group.

Since pCO2 was unchanged during the study, alveolar hypoventilation does not explain this observation. Likewise, no alterations of lung volumes, airflow or DLCO and DLNO were observed in either group. Possibly, RIP transiently affected regional pulmonary blood flow and the relationship between alveolar ventilation and perfusion due to vasoactive effects. However, measurement of ventilation-perfusion (VA/Q) distribution e.g. by multiple inert gas elimination technique (MIGET) or single photon emission tomography (SPECT) was not possible for ethical and technical reasons in our study (53, 54).

Different studies have assessed pulmonary effects of RIP with conflicting results. Foster et al. found that a 90 minutes hypoxemic challenge using nitrogen-enriched gas mixture titrated to an FIO2 of 0.13 significantly increased resting pulmonary arterial pressures (55). This effect was attenuated by RIP suggesting an impact on HPV. These findings were recently confirmed by Kim et al. in healthy volunteers randomized to either RIP (n=8) or control (n=8) (56). RIP decreased systolic and mean pulmonary arterial pressure and improved breathing efficiency (VE/VCO2) and end-tidal pCO2, which was attributed to effects on the NO-cGMP pathway. However, no blood gas data were presented in both studies. Berger et al. studied healthy volunteers subjected to 18 hours of normobaric hypoxia induced by breathing a gas mixture containing 12% oxygen (57). In the RIP group, biomarkers of oxidative stress including ascorbate radicals, oxidized SH groups and electron paramagnetic resonance signal intensity were significantly ameliorated after five hours. These effects were no longer apparent after 18 hours of hypoxia suggesting that late pulmonary effects of RIP might differ from other target organs. In contrast, Rieger et al. failed to demonstrate changes of brachial artery flow-mediated dilation (FMD), pulmonary artery systolic pressure (PASP) or internal carotid artery (ICA) flow at sea level in healthy volunteers subjected to normoxia and isocapnic hypoxia (50). At high-altitude, hypoxic ventilatory response increased 24 hours after RIP without changes of FMD, PASP or ICA flow. Sabino-Carvalho et al. described no changes induced by RIP in pulmonary ventilation and gas exchange after endurance exercise in trained runners, suggesting that the endothelial function in athletes is already preconditioned by regular training (58). The utility of RIP during acute and chronic hypoxia or during endurance exercise remains to be shown.

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The Effects of RIP on Oxygenation in Pigs In the Studies II and III, oxygenation was increased in the RIP group after OLV agreeing with previous studies (59). This effect can be related to the modulation of regional lung perfusion, resulting in improved ventilation-perfusion matching. Persistent hyperperfusion of the ventilated lung after OLV has been demonstrated by single photon emission tomography (19). Therefore, RIP may affect pulmonary vasoconstriction in the previously collapsed lung and thereby improve oxygenation.

The Effects of RIP on Alveolar Damage MV including two hours of OLV results in significantly increased DAD scores, which confirms earlier histologic studies using a corresponding experimental protocol (15). In addition, clinical studies revealed that lung resection surgery is associated with increased markers of oxidative stress and inflammation (26-28). Previous data obtained by bronchoscopic microsampling from each lung showed that IL-1β, IL-6, and IL-8 remained significantly increased both in the ventilated and in the non-ventilated lung at the end of lung resection surgery (29). Moreover, the production and release of superoxide anions and cytokines may be induced by HPV in the collapsed lung, and OLV has been shown to increase concentrations of 8-isoprostane, NO2

- and NO3- and H2O2 in exhaled breath condensate (30). However, in the

present study, RIP was not associated with different DAD scores, indicating that early alveolar damage is not prevented or attenuated by RIP. Histologic analysis revealed slightly higher degrees of microhemorrhage and alveolar edema in the ventilated lung suggesting that RIP may even increase pulmonary capillary perfusion.

The Effects of RIP on the Immune Response Protective ventilatory settings have been established to prevent the lungs from mechanical damage provoked by shear stress and increased airway pressures. But still, chemical issues might also be worthwhile to look at to attenuate the release of pro-inflammatory cytokines and support anti-inflammatory pathways. In this context, RIP is one of the most promising features (60). However, the mechanisms of RIP are still not fully elucidated, but likely act via multiple pathways. Short episodes of ischemia applied to one limb activate kinases and transcription factors (61) which are released into the circulation, enabling remote organs to resist ischemia at a later point in time (17). The expression of anti-oxidative enzymes and the suppression of ROS seem to play a role as well as the transcription and expression of anti-inflammatory

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mediators, which activate intracellular pathways after binding to their receptor (62), leading to the opening of KATP-channels and preventing permeability-pores from opening, thus protecting the mitochondria and lowering the electrical potential of the membrane (63). Eventually, the cells will activate their self-repair-mechanisms and improve their survival, preventing ischemic injury and thus inflammation and apoptosis (4).

The wet lung / dry lung weight ratio is reduced after RIP applied to the liver in a rat model, indicating that RIP has an effect on the development of edema (64). However, this is not exactly in line with our results, as we have found that alveolar edema is increased after RIP. This might hint that the site of RIP plays a role as well, as we performed RIP on the hind limb and not on an intestinal organ. Moreover, our model opens an exciting field for further studies that will analyze pulmonary edema formation but also may include mRNA-expression of different mediators.

Additional RIP-mechanisms might include cytokines and damage-associated molecular pattern (DAMP) molecules, which are intracellular danger signals such as high-mobility group box 1 (HMGB1), and a multigenic family of calcium modulated proteins involved in cellular regulatory activity (S100A8/A9) (64). These bind to toll-like receptors (TLR) or receptors for advanced glycation endproducts (RAGE) and promote nuclear factor-κB (NF-κB) signaling and expression of cytokines functioning through their respective receptors (65). NF-κB-dependent proinflammatory cytokines upregulate the expression of DAMPs and RAGE, thus leading to a pathological cycle of inflammation (66). The downstream effects on macrophages were assessed in this study, using interleukins and TNF-α as surrogate markers for HMGB1 and S100, which are fairly easy to measure in porcine samples. In fact, the concentrations of pro-inflammatory TNF-α and of alveolar protein were lower in the ventilated lung in the RIP group as compared to the control group, which may suggest an anti-inflammatory effect of RIP. Thus, our findings agree with a clinical study on thoracic surgical patients. As IL-8 and TNF-α develop differently, it might be that the effects of RIP on the lungs are not consistent for all cytokines or that they rise and decline at different time points.

It is difficult to assess potential differences of pulmonary cytokines in a clinical setting. The differences of IL-8 in the ventilated side in the RIP group and unchanged IL-8 serum concentrations may indicate that RIP has a direct effect on the pulmonary immune system. In healthy volunteers, RIP attenuated normal hypoxic increase in PASP, possibly by attenuation of HPV (55). The production of ROS is increased in hypoxic tissue in complex I and III of the mitochondrial chain, which may augment HPV (67). Thus, limb RIP ameliorates oxidative lung damage but may aggravate hyperperfusion of the ventilated lung during and after OLV (59).

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The Effects of RIP on BAL Fluid The leukocyte counts in BAL revealed that RIP markedly reduced alveolar sequestration of granulocytes. In contrast, the IL-8 release was high in both the control and the RIP group. However, the pathways regulating recruitment of circulating neutrophils and transmigration into the alveolar compartment are different from the pathways regulating IL-8 release (68). The inflammatory response to lung resection and OLV including neutrophil sequestration may reach a maximum in the postoperative course (69) but the present study was not designed to look at a prolonged time period after OLV.

Granulocytes are the main source of TNF-α and were decreased in BAL fluid after RIP. Therefore, RIP may indirectly affect pulmonary immune function by attenuating neutrophil sequestration. It is important to notice that the anesthetics might influence the effect of RIP. Thus, highly lipid-soluble drugs like propofol, as we used, may influence e.g. neutrophil activation and inhibit respiratory burst in the lung. Volatile anesthetics seem to be more immune depressive. In fact, we have previously demonstrated in a number of clinical and experimental studies that the volatile anesthetics desflurane and sevoflurane suppress the pro-inflammatory response in the ventilated lung after OLV more than propofol does (22, 70). In contrast, propofol does not exert this alleviating effect on alveolar cytokines. Consequently, a recent clinical study provided clear evidence for the efficacy of limb RIP even under propofol–remifentanil anesthesia (40).

The Effects of RIP on Exhaled NO Nitric oxide is known to be one marker for inflammation when assessed in the exhaled breath (71). The measurement of exhaled NO has evolved as a non-invasive method to evaluate airway inflammation. In fact, exhaled NO is an established tool in the diagnosis and management of bronchial asthma which is characterized by a chronic state of lung inflammation (72). Especially in asthmatic patients, exhaled NO correlates with airway eosinophilia (73, 74). NO is produced by several NO synthases (NOS; neuronal, inducible, endothelial) (47, 75, 76) and has numerous physiological effects (77). Endothelial NOS (eNOS) originates from endothelial cells (78) and is stimulated by shear stresses. It results in vasodilation via cGMP production. cGMP acts as second messenger and activates proteinkinase C (PKC) which decreases intracellular calcium and reduces vascular smooth muscle tone, leading thus to vasodilation (79, 80). NO is produced by endothelial cells all over the body as well as in the airways and contributes to the fraction of NO in the exhaled air (81).

Alterations in airway caliber seem to impact on the fraction of exhaled NO, bronchoconstriction lowering the levels of expired NO (82-84). In a clinical

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study conducted on asthmatic patients (85) expired NO decreased after the administration of metacholine. This was due to a reduction of NO flux through the airway wall and to a diminished diffusing capacity for NO as the luminal airway surface decreased. A local obstruction might also be involved in the reduction of exhaled NO so that NO is trapped in the post-obstructed areas of the lungs and cannot be expired. Against the background of this study, the decline of exhaled NO during and after OLV in the control group can be interpreted as a result of vasoconstriction. The drop in airway caliber induced a drop in exhaled NO in the piglets.

Cattoni’s findings (85) are in line with the results of an experiment carried out in rats (86). The animals were rendered septic and compared to controls. Among the septic animals, the fraction of exhaled NO declined. As the enzymes that catalyze the production of NO are oxygen dependent, this decline in expired NO was attributed to hypoxia in the septic lungs. The authors conclude that NO does not have a major role in HPV but that – on the other hand – hypoxia suppresses the enzymes that produce NO and thus exhaled NO is reduced. Therefore, exhaled NO might evolve as a marker for HPV and for ventilation-perfusion-matching, rather than NO leading to and reinforcing HPV. These findings are in agreement with our results in that exhaled NO is reduced during and after OLV, possibly due to HPV. In accordance with Hakim et al., it can be postulated that NO in itself does not contribute to HPV but rather results out of it. Therefore, exhaled NO may rather be a consequence of HPV and not its cause. Exhaled NO also declines in the RIP group during OLV, and this is in line with our findings (5) that RIP might attenuate HPV and eventually lead to an improvement in ventilation-perfusion matching.

Karamaoun and co-workers applied a mathematical model to the production and the transport of NO in the lungs (87). They confirmed that NO flux from the endothelial cell to the airway space is highly dependent on airway caliber. Exhaled NO is therefore not only a marker for inflammation but also for airway caliber changes. Their results indicate that NO flux and exhaled NO decline when the caliber drops. If a fluid layer is added to the airway lumen, NO flux and the fraction of exhaled NO will further go down. Our results support this in that exhaled NO declines in the control group during and after OLV. OLV has been shown to induce a pro-inflammatory state in the lung (32). We have demonstrated that the inflammatory response to OLV can be attenuated by RIP (5). Exhaled NO did not decrease during and after OLV in the RIP group, whereas hemodynamic and gas exchange variables were comparable between both groups. Whether this effect originates from alterations of inflammation and NO production in the conducting airways, pulmonary vessels or both remains to be shown. Likewise, it cannot be stated if the attenuation of the decline in exhaled NO after RIP is beneficial or not. It is rather a consequence of that the airway caliber does not drop. The mechanisms are not yet elucidated.

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Limitations of the Clinical Study There are different limitations in Study I, including the model per se, the sample size of the study and the control group, a fixed setup of RIP and the short post-interventional observation period. The optimal magnitude of the ischemic stimulus for RIP is unclear. Possibly, a dose-response relationship exists, and prolonged or more severe episodes of ischemia may induce additional effects on different target organs (88). However, the study protocol is closely related to different studies on RIP in healthy volunteers (50, 89) and cardiothoracic patients (59). It was not the principal aim of the present study to demonstrate lung protective effects but to analyze potential effects of RIP on spirometry variables and gas exchange in healthy, awake and spontaneously breathing subjects.

Limitations of the Experimental Studies There are limitations in the current Studies II and III, including the restrictions of the animal model per se, the sample size of the study group, the fixed ventilation setup for mechanical ventilation and the short post-interventional observation period. Thus, prolonged effects of RIP could not be assessed. The intention of this experiment was to induce a significant alveolar injury by OLV, which was stable and reproducible. Despite hemodynamic parameters were similar in both groups, increased MAP in the RIP group can be related to high individual scatter. Thus, the applied ventilation settings and time schedule may cover a multitude of clinical procedures. This study was closely related to previous experiments on OLV-induced pulmonary changes in pigs (15, 19). By using the established protocol, it becomes possible to validate and compare the present data to previous results. Moreover, it was not the principal aim of the present study to demonstrate effects of protective ventilation but to analyze potential effects of RIP in an animal model of OLV. Whether RIP further augments a lung protective approach in thoracic surgical patients can be addressed in future studies.

Study IV has several limitations. We have analyzed juvenile piglets and assessed the effects of RIP on healthy lungs. Pulmonary and cardiocirculatory physiology in pigs is different from human patients. We aimed at comparing the present results to our earlier findings from comparable animal experiments (3, 5) in that we extended the time of the protocol that we used earlier. However, there are differences in the study protocol; especially, we did not perform regular BAL. We abstained from these in order not to harm the lung any further and thus to avoid any possible confounders. The data on exhaled NO after the administration of RIP before OLV is novel and open the field for further experiments to assess the impact of RIP on ventilation-perfusion-matching and its mechanisms.

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CONCLUSIONS

The overall objective of the present studies was to demonstrate the effects of RIP on gas exchange and on the immune response. The aim of Study I was to assess the consequences of RIP in spontaneously breathing, healthy volunteers. All other confounders that are encountered in a clinical setting – pre-existing disease and drug intake, anesthesia including mechanical ventilation, surgery – have been eliminated in the protocol so that the focus was exclusively on the effects of RIP which was the only intervention the subjects were submitted to. In summary, RIP had no effect on lung volumes, diffusion capacity or airflow in healthy volunteers. Oxygenation was slightly impaired one hour after RIP, which may be explained by transient alterations of ventilation-perfusion-relationships, but no effect was observed after 24 hours.

In conclusion of the Studies II and III, the pulmonary effects of RIP in an experimental model of mechanical ventilation may include a decreased pro-inflammatory response and alveolar leukocyte sequestration into both lungs. The cytokine levels in serum and in pulmonary tissue show different time patterns for each protein. It cannot be said whether RIP in itself is beneficial to the lung or not, as the overall damage score remains unchanged and two aspects of it – microhemorrhage and alveolar edema – are even increased after RIP. This suggests that pulmonary capillary perfusion is affected which confirms the results of Study I.

In conclusion of Study IV, the data confirm that oxygenation improves after two hours of OLV in the RIP group and the decline of exhaled NO after OLV is discontinued in the RIP group. Considering that exhaled NO is a marker for caliber changes in the airways, this indicates that RIP has a mitigating effect on HPV and thus on ventilation-perfusion-matching.

All the results from the clinical and the experimental studies hint at RIP influencing the ventilation-perfusion-matching. If this effect is brought about by the immune response is still not elucidated and opens the field for further research. Even though promising, it is unlikely that RIP will be established in clinical practice as a tool for improving patients’ outcome.

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ACKNOWLEDGEMENTS

First and foremost, I am hugely indebted to my mentor and supervisor Thomas Hachenberg. He has just the right mix of discipline and indulgence to get me past the finishing line. He opened the new horizon of science to me and made all the days of doubt and despair worthwhile.

I am grateful to Göran Hedenstierna for his generous invitation to the lab that carries his name, for his kindness and patience in supervising my work, and for his sparkling motivation.

My sincere thanks to Anders Larsson, my tutor, for all his unerringly useful notes and his great support. His open mind inspired me, and his advice guided me to ask the right questions at the right time.

My colleague and above all my dear friend Thomas Schilling provided wonderful support throughout the process and made it beautiful. Heartfelt thanks to him.

Eva-Maria Hedin made me feel at home in Uppsala. She is my trusted and incomparable friend, and I am grateful for her unparalleled energy and her wise and warm counsel in all aspects.

I wish to express my deepest appreciation to the entire team at Hedenstierna Laboratoriet, especially to Agneta Roneus, Kerstin Ahlgren, Monika Hall, Mariette Andersson, Maria Swälas. The lab is a magic and special place because of all of them, their kind support and creativity.

Very special thanks go to my colleagues, fellow researchers and friends Mariangela Pellegrini and Gaetano Perchiazzi. We had stimulating discussions about science and things that matter.

My colleagues in Magdeburg, especially those from cardio-thoracic anesthesia, did without me ever so often throughout the last three years, and I am grateful to them.

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