Chronic and acute stress in atherosclerosis : the role of endothelial function and arterial elasticity

Atherosclerosis is the main underlying pathology of coronary heart disease. Coronary heart disease is a serious health problem in Finland, and it is the leading cause of morbidity and mortality in industrialized countries. Psychological stress correlates with coronary heart disease events – myocardial infarction and sudden death, which are the most common clinical syndromes of atherosclerotic narrowing of arteries. The present series of studies examines the interaction between stress and endothelial function in relation to atherosclerosis. The study also aims to give new information on the mechanisms through which stress has its effect on atherosclerosis progression, focusing on possible relations between psychological stress and the functioning of the endothelium. Our project is based on data from one of the largest national epidemiological studies, the Cardiovascular Risk in Young Finns study, which has monitored the development of risk factors for coronary heart disease in 3596 young adults since 1980. The present study combines experimental stress research with epidemiology and uses an advanced method for examining atherosclerosis development in healthy subjects (intima-media thickness ultrasound measurement). The physiological parameters used were heart rate, respiratory sinus arrhythmia and pre-ejection period. Chronic stress was assessed by vital exhaustion. The ultrasound measurements that served as the indexes of preclinical atherosclerosis were carotid intima-media thickness, brachial flow-mediated dilatation and carotid artery compliance. The effects of cardiovascular risk factors found to be important were taken into account: serum cholesterols level, triglyceride level, serum insulin level and systolic and diastolic blood pressure. There were 69, 1596, 81 and 1721 participants in studies I-IV, respectively. The results showed that both chronic and acute stress may exert an effect on atherosclerosis in subjects with impaired endothelial responses. The findings are consistent with the idea that risk factors are more harmful if the endothelium is not working properly. Chronic stress was found to be a risk if it has resulted in ineffective cardiac stress reactivity or delayed recovery. Men were shown to be at increased risk for atherosclerotic progression in early life, which suggests men’s decreased stress coping ability in relation to stressful psychosocial coronary risk factors. Autonomic imbalance may be the common mechanism of the stress influence on atherosclerosis development. The results of the present study contain background information for the identification the first stages of atherosclerosis, and they may be useful for preventive medicine programs for young adults and could help to improve cardiovascular health in Finland as well as in other countries.

Microclimate and gas emissions in dairy buildings : Instrumentation, theory and measurements

The aim of this thesis was to develop measurement techniques and systems for measuring air quality and to provide information about air quality conditions and the amount of gaseous emissions from semi-insulated and uninsulated dairy buildings in Finland and Estonia. Specialization and intensification in livestock farming, such as in dairy production, is usually accompanied by an increase in concentrated environmental emissions. In addition to high moisture, the presence of dust and corrosive gases, and widely varying gas concentrations in dairy buildings, Finland and Estonia experience winter temperatures reaching below -40 ºC and summer temperatures above +30 ºC. The adaptation of new technologies for long-term air quality monitoring and measurement remains relatively uncommon in dairy buildings because the construction and maintenance of accurate monitoring systems for long-term use are too expensive for the average dairy farmer to afford. Though the documentation of accurate air quality measurement systems intended mainly for research purposes have been made in the past, standardised methods and the documentation of affordable systems and simple methods for performing air quality and emissions measurements in dairy buildings are unavailable. In this study, we built three measurement systems: 1) a Stationary system with integrated affordable sensors for on-site measurements, 2) a Wireless system with affordable sensors for off-site measurements, and 3) a Mobile system consisting of expensive and accurate sensors for measuring air quality. In addition to assessing existing methods, we developed simplified methods for measuring ventilation and emission rates in dairy buildings. The three measurement systems were successfully used to measure air quality in uninsulated, semi-insulated, and fully-insulated dairy buildings between the years 2005 and 2007. When carefully calibrated, the affordable sensors in the systems gave reasonably accurate readings. The spatial air quality survey showed high variation in microclimate conditions in the dairy buildings measured. The average indoor air concentration for carbon dioxide was 950 ppm, for ammonia 5 ppm, for methane 48 ppm, for relative humidity 70%, and for inside air velocity 0.2 m/s. The average winter and summer indoor temperatures during the measurement period were -7º C and +24 ºC for the uninsulated, +3 ºC and +20 ºC for the semi-insulated and +10 ºC and +25 ºC for the fully-insulated dairy buildings. The measurement results showed that the uninsulated dairy buildings had lower indoor gas concentrations and emissions compared to fully insulated buildings. Although occasionally exceeded, the ventilation rates and average indoor air quality in the dairy buildings were largely within recommended limits. We assessed the traditional heat balance, moisture balance, carbon dioxide balance and direct airflow methods for estimating ventilation rates. The direct velocity measurement for the estimation of ventilation rate proved to be impractical for naturally ventilated buildings. Two methods were developed for estimating ventilation rates. The first method is applicable in buildings in which the ventilation can be stopped or completely closed. The second method is useful in naturally ventilated buildings with large openings and high ventilation rates where spatial gas concentrations are heterogeneously distributed. The two traditional methods (carbon dioxide and methane balances), and two newly developed methods (theoretical modelling using Fick s law and boundary layer theory, and the recirculation flux-chamber technique) were used to estimate ammonia emissions from the dairy buildings. Using the traditional carbon dioxide balance method, ammonia emissions per cow from the dairy buildings ranged from 7 g day-1 to 35 g day-1, and methane emissions per cow ranged from 96 g day-1 to 348 g day-1. The developed methods proved to be as equally accurate as the traditional methods. Variation between the mean emissions estimated with the traditional and the developed methods was less than 20%. The developed modelling procedure provided sound framework for examining the impact of production systems on ammonia emissions in dairy buildings.

Behavioural and physiological responses to predators of captive-bred Arctic charr: significance of genetics, learning and ontogeny

Predation forms one of the main selective forces in nature and in a vast number of prey species the behavioural responses form the main way to avoid predation. World wide numerous captive breeding programs are used to produce fish and other animal species for conservational reintroductions. However, rearing animals in the absence of predators in captivity has been shown to weaken their predator avoidance skills and lead to behavioural divergence between wild and captive-bred populations. In my thesis I studied the effects of predator odour exposures on antipredator behavioural and physiological responses of captive reared Saimaa Arctic charr. This charr population is the most endangered fish population in Finland and a sample of the remaining population has been taken to captive breeding and used for an extensive reintroduction program. Lowered responsiveness to predators is probably one of the major reasons for the poor survival probability of the charr after release into the wild. The main aims of my thesis were to explore the reasons for behavioural phenotypic variation in this charr population and whether naïve charr young could be trained to recognise their natural predators. The predator species in my thesis were burbot (Lota lota) and pikeperch (Sander lucioperca). In my thesis I showed that the captive-bred charr responded to chemical cues from burbot and pikeperch, but the magnitude of responses was linked to the predator species. The burbot odour increased the spatial odour avoidance of the charr young. On the other hand, in the pikeperch treatment charr reduced their relative swimming activity and tended to show more freezing behaviour relative to the burbot treatment. It seems evident that these different responses are related to the different hunting tactics of predator species. Furthermore, I detected wide between-family differences in antipredator responsiveness (i.e. inherited variation in antipredator behaviours) in this captive stock. Detected differences were greater in the response towards burbot than towards pikeperch. These results, in addition to predator-specific antipredator responses, suggest that there is a clear inherited component in antipredator responsiveness in Saimaa charr population and that the detected inherited differences could explain a part of the behavioural phenotypic variation in this population. In my thesis I also found out that both social learning and direct exposure to live predators enhance the antipredator responsiveness of charr young. In addition, I obtained indications that predator odour exposures (i.e. life-skills training) in alevin and fry stages can fine-tune the innate antipredator responsiveness of charr. Thus, all these methods have the potential to enhance the innate antipredator responsiveness of naïve charr young, possibly also improving the post-release survival of these trained individuals in the wild. However, the next logical phase would be to carry out large scale survival studies in the wild to test this hypothesis. Finally, the results of my thesis emphasize that possible long-term life-skills training methods should take into account not only the behavioural but also the physiological effects of training.

Fluorescence properties of Baltic Sea phytoplankton

To obtain data on phytoplankton dynamics with improved spatial and temporal resolution, and at reduced cost, traditional phytoplankton monitoring methods have been supplemented with optical approaches. In this thesis, I have explored various fluorescence-based techniques for detection of phytoplankton abundance, taxonomy and physiology in the Baltic Sea. In algal cultures used in this thesis, the availability of nitrogen and light conditions caused changes in pigmentation, and consequently in light absorption and fluorescence properties of cells. In the Baltic Sea, physical environmental factors (e.g. mixing depth, irradiance and temperature) and related seasonal succession in the phytoplankton community explained a large part of the seasonal variability in the magnitude and shape of Chlorophyll a (Chla)-specific absorption. The variability in Chla-specific fluorescence was related to the abundance of cyanobacteria, the size structure of the phytoplankton community, and absorption characteristics of phytoplankton. Cyanobacteria show very low Chla-specific fluorescence. In the presence of eukaryotic species, Chla fluorescence describes poorly cyanobacteria. During cyanobacterial bloom in the Baltic Sea, phycocyanin fluorescence explained large part of the variability in Chla concentrations. Thus, both Chla and phycocyanin fluorescence were required to predict Chla concentration. Phycobilins are major light harvesting pigments for cyanobacteria. In the open Baltic Sea, small picoplanktonic cyanobacteria were the main source of phycoerythrin fluorescence and absorption signal. Large filamentous cyanobacteria, forming harmful blooms, were the main source of the phycocyanin fluorescence signal and typically their biomass and phycocyanin fluorescence were linearly related. Using phycocyanin fluorescence, dynamics of cyanobacterial blooms can be detected at high spatial and seasonal resolution not possible with other methods. Various taxonomic phytoplankton pigment groups can be separated by spectral fluorescence. I compared multivariate calibration methods for the retrieval of phytoplankton biomass in different taxonomic groups. Partial least squares regression method gave the closest predictions for all taxonomic groups, and the accuracy was adequate for phytoplankton bloom detection. Variable fluorescence has been proposed as a tool to study the physiological state of phytoplankton. My results from the Baltic Sea emphasize that variable fluorescence alone cannot be used to detect nutrient limitation of phytoplankton. However, when combined with experiments with active nutrient manipulation, and other nutrient limitation indices, variable fluorescence provided valuable information on the physiological responses of the phytoplankton community. This thesis found a severe limitation of a commercial fast repetition rate fluorometer, which couldn t detect the variable fluorescence of phycoerythrin-lacking cyanobacteria. For these species, the Photosystem II absorption of blue light is very low, and fluorometer excitation light did not saturate Photosystem II during a measurement. This thesis encourages the use of various in vivo fluorescence methods for the detection of bulk phytoplankton biomass, biomass of cyanobacteria, chemotaxonomy of phytoplankton community, and phytoplankton physiology. Fluorescence methods can support traditional phytoplankton monitoring by providing continuous measurements of phytoplankton, and thereby strengthen the understanding of the links between biological, chemical and physical processes in aquatic ecosystems.

Preoperative visual acuity of cataract patients. Repeatability of visual acuity and refractive error measurements in clinical settings

Visual acuities at the time of referral and on the day before surgery were compared in 124 patients operated on for cataract in Vaasa Central Hospital, Finland. Preoperative visual acuity and the occurrence of ocular and general disease were compared in samples of consecutive cataract extractions performed in 1982, 1985, 1990, 1995 and 2000 in two hospitals in the Vaasa region in Finland. The repeatability and standard deviation of random measurement error in visual acuity and refractive error determination in a clinical environment in cataractous, pseudophakic and healthy eyes were estimated by re-examining visual acuity and refractive error of patients referred to cataract surgery or consultation by ophthalmic professionals. Altogether 99 eyes of 99 persons (41 cataractous, 36 pseudophakic and 22 healthy eyes) with a visual acuity range of Snellen 0.3 to 1.3 (0.52 to -0.11 logMAR) were examined. During an average waiting time of 13 months, visual acuity in the study eye decreased from 0.68 logMAR to 0.96 logMAR (from 0.2 to 0.1 in Snellen decimal values). The average decrease in vision was 0.27 logMAR per year. In the fastest quartile, visual acuity change per year was 0.75 logMAR, and in the second fastest 0.29 logMAR, the third and fourth quartiles were virtually unaffected. From 1982 to 2000, the incidence of cataract surgery increased from 1.0 to 7.2 operations per 1000 inhabitants per year in the Vaasa region. The average preoperative visual acuity in the operated eye increased by 0.85 logMAR (in decimal values from 0.03to 0.2) and in the better eye 0.27 logMAR (in decimal values from 0.23 to 0.43) over this period. The proportion of patients profoundly visually handicapped (VA in the better eye <0.1) before the operation fell from 15% to 4%, and that of patients less profoundly visually handicapped (VA in the better eye 0.1 to <0.3) from 47% to 15%. The repeatability visual acuity measurement estimated as a coefficient of repeatability for all 99 eyes was ±0.18 logMAR, and the standard deviation of measurement error was 0.06 logMAR. Eyes with the lowest visual acuity (0.3-0.45) had the largest variability, the coefficient of repeatability values being ±0.24 logMAR and eyes with a visual acuity of 0.7 or better had the smallest, ±0.12 logMAR. The repeatability of refractive error measurement was studied in the same patient material as the repeatability of visual acuity. Differences between measurements 1 and 2 were calculated as three-dimensional vector values and spherical equivalents and expressed by coefficients of repeatability. Coefficients of repeatability for all eyes for vertical, torsional and horisontal vectors were ±0.74D, ±0.34D and ±0.93D, respectively, and for spherical equivalent for all eyes ±0.74D. Eyes with lower visual acuity (0.3-0.45) had larger variability in vector and spherical equivalent values (±1.14), but the difference between visual acuity groups was not statistically significant. The difference in the mean defocus equivalent between measurements 1 and 2 was, however, significantly greater in the lower visual acuity group. If a change of ±0.5D (measured in defocus equivalents) is accepted as a basis for change of spectacles for eyes with good vision, the basis for eyes in the visual acuity range of 0.3 - 0.65 would be ±1D. Differences in repeated visual acuity measurements are partly explained by errors in refractive error measurements.

Measurements of adequacy of anesthesia and level of consciousness during surgery and intensive care

The adequacy of anesthesia has been studied since the introduction of balanced general anesthesia. Commercial monitors based on electroencephalographic (EEG) signal analysis have been available for monitoring the hypnotic component of anesthesia from the beginning of the 1990s. Monitors measuring the depth of anesthesia assess the cortical function of the brain, and have gained acceptance during surgical anesthesia with most of the anesthetic agents used. However, due to frequent artifacts, they are considered unsuitable for monitoring consciousness in intensive care patients. The assessment of analgesia is one of the cornerstones of general anesthesia. Prolonged surgical stress may lead to increased morbidity and delayed postoperative recovery. However, no validated monitoring method is currently available for evaluating analgesia during general anesthesia. Awareness during anesthesia is caused by an inadequate level of hypnosis. This rare but severe complication of general anesthesia may lead to marked emotional stress and possibly posttraumatic stress disorder. In the present series of studies, the incidence of awareness and recall during outpatient anesthesia was evaluated and compared with that of in inpatient anesthesia. A total of 1500 outpatients and 2343 inpatients underwent a structured interview. Clear intraoperative recollections were rare the incidence being 0.07% in outpatients and 0.13% in inpatients. No significant differences emerged between outpatients and inpatients. However, significantly smaller doses of sevoflurane were administered to outpatients with awareness than those without recollections (p<0.05). EEG artifacts in 16 brain-dead organ donors were evaluated during organ harvest surgery in a prospective, open, nonselective study. The source of the frontotemporal biosignals in brain-dead subjects was studied, and the resistance of bispectral index (BIS) and Entropy to the signal artifacts was compared. The hypothesis was that in brain-dead subjects, most of the biosignals recorded from the forehead would consist of artifacts. The original EEG was recorded and State Entropy (SE), Response Entropy (RE), and BIS were calculated and monitored during solid organ harvest. SE differed from zero (inactive EEG) in 28%, RE in 29%, and BIS in 68% of the total recording time (p<0.0001 for all). The median values during the operation were SE 0.0, RE 0.0, and BIS 3.0. In four of the 16 organ donors, EEG was not inactive, and unphysiologically distributed, nonreactive rhythmic theta activity was present in the original EEG signal. After the results from subjects with persistent residual EEG activity were excluded, SE, RE, and BIS differed from zero in 17%, 18%, and 62% of the recorded time, respectively (p<0.0001 for all). Due to various artifacts, the highest readings in all indices were recorded without neuromuscular blockade. The main sources of artifacts were electrocauterization, electromyography (EMG), 50-Hz artifact, handling of the donor, ballistocardiography, and electrocardiography. In a prospective, randomized study of 26 patients, the ability of Surgical Stress Index (SSI) to differentiate patients with two clinically different analgesic levels during shoulder surgery was evaluated. SSI values were lower in patients with an interscalene brachial plexus block than in patients without an additional plexus block. In all patients, anesthesia was maintained with desflurane, the concentration of which was targeted to maintain SE at 50. Increased blood pressure or heart rate (HR), movement, and coughing were considered signs of intraoperative nociception and treated with alfentanil. Photoplethysmographic waveforms were collected from the contralateral arm to the operated side, and SSI was calculated offline. Two minutes after skin incision, SSI was not increased in the brachial plexus block group and was lower (38 ± 13) than in the control group (58 ± 13, p<0.005). Among the controls, one minute prior to alfentanil administration, SSI value was higher than during periods of adequate antinociception, 59 ± 11 vs. 39 ± 12 (p<0.01). The total cumulative need for alfentanil was higher in controls (2.7 ± 1.2 mg) than in the brachial plexus block group (1.6 ± 0.5 mg, p=0.008). Tetanic stimulation to the ulnar region of the hand increased SSI significantly only among patients with a brachial plexus block not covering the site of stimulation. Prognostic value of EEG-derived indices was evaluated and compared with Transcranial Doppler Ultrasonography (TCD), serum neuron-specific enolase (NSE) and S-100B after cardiac arrest. Thirty patients resuscitated from out-of-hospital arrest and treated with induced mild hypothermia for 24 h were included. Original EEG signal was recorded, and burst suppression ratio (BSR), RE, SE, and wavelet subband entropy (WSE) were calculated. Neurological outcome during the six-month period after arrest was assessed with the Glasgow-Pittsburgh Cerebral Performance Categories (CPC). Twenty patients had a CPC of 1-2, one patient had a CPC of 3, and nine patients died (CPC 5). BSR, RE, and SE differed between good (CPC 1-2) and poor (CPC 3-5) outcome groups (p=0.011, p=0.011, p=0.008, respectively) during the first 24 h after arrest. WSE was borderline higher in the good outcome group between 24 and 48 h after arrest (p=0.050). All patients with status epilepticus died, and their WSE values were lower (p=0.022). S-100B was lower in the good outcome group upon arrival at the intensive care unit (p=0.010). After hypothermia treatment, NSE and S-100B values were lower (p=0.002 for both) in the good outcome group. The pulsatile index was also lower in the good outcome group (p=0.004). In conclusion, the incidence of awareness in outpatient anesthesia did not differ from that in inpatient anesthesia. Outpatients are not at increased risk for intraoperative awareness relative to inpatients undergoing general anesthesia. SE, RE, and BIS showed non-zero values that normally indicate cortical neuronal function, but were in these subjects mostly due to artifacts after clinical brain death diagnosis. Entropy was more resistant to artifacts than BIS. During general anesthesia and surgery, SSI values were lower in patients with interscalene brachial plexus block covering the sites of nociceptive stimuli. In detecting nociceptive stimuli, SSI performed better than HR, blood pressure, or RE. BSR, RE, and SE differed between the good and poor neurological outcome groups during the first 24 h after cardiac arrest, and they may be an aid in differentiating patients with good neurological outcomes from those with poor outcomes after out-of-hospital cardiac arrest.

Balance of growth factors in the human perinatal lung : Implications for physiological lung development and link to bronchopulmonary dysplasia

The aims of this Thesis was to evaluate the role of proangiogenic placental growth factor (PlGF), antiangiogenic endostatin and lymphangiogenic vascular endothelial growth factor (VEGF) -C as well as the receptors vascular endothelial growth factor receptor (VEGFR) -2 and VEGFR-3 during lung development and in development of lung injury in preterm infants. The studied growth factors were selected due to a close relationship with VEGF-A; a proangiogenic growth factor important in normal lung angiogenesis and lung injury in preterm infants. The thesis study consists of three analyses. I: Lung samples from fetuses, preterm and term infants without lung injury, as well as preterm infants with acute and chronic lung injury were stained by immunohistochemistry for PlGF, endostatin, VEGF-C, VEGFR-2 and VEGFR-3. II: Tracheal aspirate fluid (TAF) was collected in the early postnatal period from a patient population consisting of 59 preterm infants, half developing bronchopulmonary dysplasia (BPD) and half without BPD. PlGF, endostatin and VEGF-C concentrations were measured by commercial enzyme-linked immunosorbent assay (ELISA). III: Cord plasma was collected from very low birth weight (VLBW) (n=92) and term (n=48) infants in conjuncture with birth and endostatin concentrations were measured by ELISA. I: All growth factors and receptors studied were consistently stained in immunohistochemistry throughout development. For endostatin in early respiratory distress syndrome (RDS), no alveolar epithelial or macrophage staining was seen, whereas in late RDS and BPD groups, both alveolar epithelium and macrophages stained positively in approximately half of the samples. VEGFR-2 staining was fairly consistent, except for the fact that capillary endothelial staining in the BPD group was significantly decreased. II: During the first postnatal week in TAF mean PlGF concentrations were stable whereas mean endostatin and VEGF-C concentrations decreased. Higher concentrations of endostatin and VEGF-C correlated with lower birth weight (BW) and associated with administration of antenatal betamethasone. Parameters reflecting prenatal lung inflammation associated with lower PlGF, endostatin and VEGF-C concentrations. A higher mean supplemental fraction of inspired oxygen during the first 2 postnatal weeks (FiO2) correlated with higher endostatin concentrations. III: Endostatin concentrations in term infants were significantly higher than in VLBW infants. In VLBW infants higher endostatin concentrations associated with the development of BPD, this association remained significant after logistic regression analysis. We conclude that PlGF, endostatin and VEGF-C all have a physiological role in the developing lung. Also, the VEGFR-2 expression profile seems to reflect the ongoing differentiation of endothelia during development. Both endostatin and VEGFR-2 seem to be important in the development of BPD. During the latter part of the first postnatal week, preterm infants developing BPD have lower concentrations of VEGF-A in TAF. Our findings of disrupted VEGFR-2 staining in capillary and septal endothelium seen in the BPD group, as well as the increase in endostatin concentrations both in TAF and cord plasma associated with BPD, seem to strengthen the notion that there is a shift in the angiogenic balance towards a more antiangiogenic environment in BPD. These findings support the vascular hypothesis of BPD.

Exploiting ground-based measurements of the Global Positioning System for numerical weather prediction

Data assimilation provides an initial atmospheric state, called the analysis, for Numerical Weather Prediction (NWP). This analysis consists of pressure, temperature, wind, and humidity on a three-dimensional NWP model grid. Data assimilation blends meteorological observations with the NWP model in a statistically optimal way. The objective of this thesis is to describe methodological development carried out in order to allow data assimilation of ground-based measurements of the Global Positioning System (GPS) into the High Resolution Limited Area Model (HIRLAM) NWP system. Geodetic processing produces observations of tropospheric delay. These observations can be processed either for vertical columns at each GPS receiver station, or for the individual propagation paths of the microwave signals. These alternative processing methods result in Zenith Total Delay (ZTD) and Slant Delay (SD) observations, respectively. ZTD and SD observations are of use in the analysis of atmospheric humidity. A method is introduced for estimation of the horizontal error covariance of ZTD observations. The method makes use of observation minus model background (OmB) sequences of ZTD and conventional observations. It is demonstrated that the ZTD observation error covariance is relatively large in station separations shorter than 200 km, but non-zero covariances also appear at considerably larger station separations. The relatively low density of radiosonde observing stations limits the ability of the proposed estimation method to resolve the shortest length-scales of error covariance. SD observations are shown to contain a statistically significant signal on the asymmetry of the atmospheric humidity field. However, the asymmetric component of SD is found to be nearly always smaller than the standard deviation of the SD observation error. SD observation modelling is described in detail, and other issues relating to SD data assimilation are also discussed. These include the determination of error statistics, the tuning of observation quality control and allowing the taking into account of local observation error correlation. The experiments made show that the data assimilation system is able to retrieve the asymmetric information content of hypothetical SD observations at a single receiver station. Moreover, the impact of real SD observations on humidity analysis is comparable to that of other observing systems.