Jori O. Ruuskanen

Expression and function of α2-adrenoceptors in zebrafish: drug effects, mRNA and receptor distributions

The α2‐adrenoceptors are G‐protein‐coupled receptors that mediate many of the physiological effects of norepinephrine and epinephrine. Mammals have three subtypes of α2‐adrenoceptors, α2A, α2B and α2C. Zebrafish, a teleost fish used widely as a model organism, has five distinct α2‐adrenoceptor genes. The zebrafish has emerged as a powerful tool to study development and genetics, with many mutations causing diseases reminiscent of human diseases. Three of the zebrafish adra2 genes code for orthologues of the mammalian α2‐adrenoceptors, while two genes code for α2Da‐ and α2Db‐ adrenoceptors, representing a duplicated, fourth α2‐adrenoceptor subtype. The three different mammalian α2‐adrenoceptor subtypes have distinct expression patterns in different organs and tissues, and mediate different physiological functions. The zebrafish α2‐adrenergic system, with five different α2‐adrenoceptors, appears more complicated. In order to deduce the physiological functions of the zebrafish α2‐adrenoceptors, we localized the expression of the five different α2‐adrenoceptor subtypes using RT–PCR, mRNA in situ hybridization, and receptor autoradiography using the radiolabelled α2‐adrenoceptor antagonist [ethyl‐3H]RS‐79948–197. Localization of the α2A‐, α2B‐ and α2C‐adrenoceptors in zebrafish shows marked conservation when compared with mammals. The zebrafish α2A, α2Da, and α2Db each partially follow the distribution pattern of the mammalian α2A: a possible indication of subfunction partitioning between these subtypes. The α2‐adrenergic system is functional in zebrafish also in vivo, as demonstrated by marked locomotor inhibition, similarly to mammals, and lightening of skin colour induced by the specific α2‐adrenoceptor agonist, dexmedetomidine. Both effects were antagonized by the specific α2‐adrenoceptor antagonist atipamezole.

Conserved structural, pharmacological and functional properties among the three human and five zebrafish α2‐adrenoceptors

1 Zebrafish has five distinct α2‐adrenoceptors. Two of these, α2Da and α2Db, represent a duplicated, fourth α2‐adrenoceptor subtype, while the others are orthologue of the human α2A‐, α2B‐ and α2C‐adrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2 The zebrafish α2‐adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist‐stimulated [35S]GTPγS binding). The affinity results were used to cluster the receptors and, separately, the ligands using both principal component analysis and binary trees. 3 The overall ligand binding characteristics, the order of potency and efficacy of the tested agonists and the G‐protein coupling of the zebrafish and human α2‐adrenoceptors, separated by ∼350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish α2‐adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40‐fold affinity differences. 4 The α2A orthologues and the zebrafish α2D duplicates clustered as close pairs, but the relationships between the orthologues of α2B and α2C were not clearly defined. Applied to the ligands, our clustering methods segregated the ligands based on their chemical structures and functional properties. As the ligand binding pockets formed by the transmembrane helices show only minor differences among the α2‐adrenoceptors, we suggest that the second extracellular loop – where significant sequence variability is located – might contribute significantly to the observed affinity differences.

The second extracellular loop of α2A-adrenoceptors contributes to the binding of yohimbine analogues

Rodent α2A‐adrenoceptors bind the classical α2‐antagonists yohimbine and rauwolscine with lower affinity than the human α2A‐adrenoceptor. A serine‐cysteine difference in the fifth transmembrane helix (TM; position 5.43) partially explains this, but all determinants of the interspecies binding selectivity are not known. Molecular models of α2A‐adrenoceptors suggest that the second extracellular loop (XL2) folds above the binding cavity and may participate in antagonist binding.

Involvement of the first transmembrane segment of human α2‐adrenoceptors in the subtype‐selective binding of chlorpromazine, spiperone and spiroxatrine

BACKGROUND AND PURPOSE Some large antagonist ligands (ARC239, chlorpromazine, prazosin, spiperone, spiroxatrine) bind to the human α2A‐adrenoceptor with 10‐ to 100‐fold lower affinity than to the α2B‐ and α2C‐adrenoceptor subtypes. Previous mutagenesis studies have not explained this subtype selectivity.

Seasonality of stroke in Finland

Abstract Introduction: The burden of stroke is increasing globally. Reports on seasonal variations in stroke occurrence are conflicting and long-term data are absent. Methods: A retrospective cohort study using discharge registry data of all acute stroke admissions in Finland during 2004–2014 for patients ≥18 years age. A total of 97,018 admissions for ischemic stroke (IS) were included, 18,252 admissions for intracerebral hemorrhage (ICH) and 11,271 admissions for subarachnoid hemorrhage (SAH). Results: The rate of IS admissions increased (p = 0.025) while SAH admission rate decreased (p < 0.0001), and ICH admission rate remained stable during the study period. The lowest seasonal admission rates were detected in summer and the highest in autumn for all stroke subtypes. Seasonal variation of IS was more pronounced in men (p = 0.020), while no sex difference was detected in ICH or SAH. The seasonal patterns of in-hospital mortality and length of stay (LOS) differed markedly by stroke subtype. Diagnoses of hypertension, atrial fibrillation, or diabetes showed no seasonality. Conclusions: All major stroke subtypes occurred most commonly in autumn and most infrequently in summer. Seasonality of in-hospital mortality and length of hospital stay appears to vary by stroke subtype. The seasonal pattern of ischemic stroke occurrence appears to have changed during the past decades. Key messages All major stroke subtypes (ischemic stroke, intracerebral hemorrhage, subarachnoid hemorrhage) occurred most frequently in autumn and least frequently in summer. Seasonal patterns of in-hospital mortality and length of stay differed markedly by stroke subtype. The seasonal pattern of ischemic stroke occurrence in Finland seems to have changed compared to 1982–1992.

Early pregnancy cerebral venous thrombosis and status epilepticus treated with levetiracetam and lacosamide throughout pregnancy.

Cerebral venous thrombosis (CVT) is an uncommon cause of stroke, accounting to less than 1% of all strokes. We describe a pregnant woman with a massive CVT in early pregnancy, complicated by status epilepticus. The mother was treated with levetiracetam, lacosamide, and enoxaparin throughout pregnancy. A male infant was born on pregnancy week 36, weighing 2.2kg. Both levetiracetam and and lacosamide were present in cord blood in levels similar to those in maternal blood. The infant was partially breast-fed and experienced poor feeding and sleepiness, starting to resolve after two first weeks. Milk samples were drawn 5 days after the delivery and a blood sample from the infant 3 days later. Lacosamide level in milk was low, resulting in an estimated relative infant dose of 1.8% of the maternal weight-adjusted daily dose in a fully breast-fed infant. This is the first case describing lacosamide use during pregnancy and lactation.

Epidemiology of Guillain-Barré syndrome in Finland 2004-2014: Epidemiology of Guillain-Barré syndrome

At total mean incidence of 0.84–1.1/100,000 the occurrence of Guillain‐Barré syndrome (GBS) is reported to be low in Finland compared to other Caucasian populations. However, a recent study from Southwestern Finland reported an incidence of 1.82/100,000 which is comparable to other Caucasian populations. We analyzed discharge data covering the years 2004 through 2014 on all neurological admissions in all Finnish university and central hospitals with a primary diagnosis of GBS. A total of 989 admissions due to GBS (917 individuals) were identified. The standardized (European population) annual incidence rate was 1.70/100,000 person‐years (95% confidence interval 1.60–1.81). GBS incidence had an increasing trend with age. The likelihood of GBS was higher among girls and adolescent women than boys and men of same age (male:female incidence rate ratio [IRR] 0.56), while in the older age groups (>19 years) the occurrence of GBS was higher among males than females (male:female IRR 1.59). The incidence of GBS remained stable during the study period. There was no seasonal variation in GBS admission frequencies (p = 0.28). No significant effect of the 2009–2010 H1N1 influenza or vaccination against it for GBS occurrence was observed. We suggest that GBS is as common, and has similar age‐distribution in Finland as in other European countries. Sex‐associated susceptibility for GBS appears to be different in children‐adolescents and adults.

No association of moon phase with stroke occurrence

ABSTRACT Stroke occurrence shows strong correlations with sleep disorders and even subtle sleep disturbances have been shown to affect ischemic stroke (IS) occurrence. Chronobiology also exerts effects, like the morning surge in IS occurrence. Lunar cycles have also been shown to affect sleep and other physiological processes, but studies on moon phases and its possible association with occurrence of stroke are rare and nonconclusive. Therefore, we studied the effects of moon phases on stroke hospitalizations and in-hospital mortality nationwide in Finland in 2004–2014. All patients aged ≥18 years with IS or intracerebral hemorrhage (ICH) as primary discharge diagnosis were included. Daily number of admissions was treated as a response variable while moon phase, year and astronomical season were independent variables in Poisson regression modeling. We found no association between moon phases and stroke occurrence. The overall occurrence rates did not vary between different moon phases for IS or ICH (p = 0.61 or higher). There were no differences between moon phases in daily admission rates among men, women, young and old patients for any of the stroke subtypes. There was no difference in in-hospital mortality with regard to moon phase for IS or ICH overall (p = 0.19 or higher), nor in subgroup analyses. There were no significant interactions between moon phase and astronomical season for stroke occurrence or in-hospital mortality. To conclude, in this over a decade-long nationwide study including a total of 46 million person years of follow-up, we found no association between moon phases and occurrence or in-hospital mortality rates of IS or intracerebral hemorrhage.

Effect of the summer holiday season on ischaemic stroke care in Finland

The standard of patient care has been suggested to suffer as freshly graduated doctors enter the workforce (the July Phenomenon in the US and the August Killing Season in the UK). The evidence on this phenomenon is conflicting and differs by medical specialties. There has been only one study investigating a possible July effect in Finland reporting no effect on the standard of care in internal medicine [1]. Reports on the subject concerning the care of acute ischaemic stroke (IS), a major cause of disability and death, are scarce with only two studies published. They were both performed in the US and reported negative results. [2,3] However, these studies have investigated a possible July effect only by comparing mortality rates. Adequate care of IS requires a high level of clinical expertise and markedly reduces the odds of poor outcome. The proportion of patients that received thrombolytic therapy and admission durations should therefore also be included in the analyses. We investigated whether the summer holiday period, when most senior neurologist are on leave and health care is staffed to a considerable degree by medical students and junior doctors with varying levels of supervision and support, affects the standard of IS care in Finland. National discharge registries were searched for all cases of IS treated in Stroke Units (SU) and neurological wards of Finnish hospitals that provide acute stroke care in 2004–2014. Data related to quality of care was analyzed and compared between July (the month with most vacation absences and, consequently, stand-ins) andNovember (no extreme weather conditions ormajor holidays and new staff that joined their institutions during the summer have found their routines). t-test and Chisquare tests were used for analyzing baseline characteristics. Length of stay (LOS, logarithm) was analyzed with linear regression and survival with Cox regression. Models were adjusted for study year, Charlson comorbity-age index (CCI), [4] sex, administration of thrombolysis and number of annual IS admissions in treating hospital (offset). There were 8058 admissions (23.6/day) that began in July and 8125 (24.6/day) that began in November. No differences were found for mean age (July: 71.8 years, November: 71.7; p=0.532) of patients, proportion of women (July: 47.8%, November: 46.9%, p = 0.272) or mean CCI (July: 5.0, November: 4.9, p = 0.344). The rate of intravenous thrombolysis administration did not differ (July: 2.7%, November:

Stroke hospitalization trends of the working-aged in Finland

Background The age-standardized incidence of stroke has decreased globally but, for reasons unknown, conflicting results have been observed regarding trend in incidence of major stroke subtypes in young adults. We studied these trends among people of working age in a population-based setting in Finland, where cardiovascular risk factor profiles have developed favorably. Methods All hospitalizations for stroke in 2004–2005 and 2013–2014 for persons 18–64 years of age were identified from a national register. The search included all hospitals that provide acute stroke care on mainland Finland. Results Hospitalizations for both intracerebral hemorrhage (ICH; -15.2%; p = 0.0008) and subarachnoid hemorrhage (SAH; -26.5%; p<0.0001) decreased overall and for both sexes separately. Concerning IS, hospitalizations decreased only for men (-6.3%; p = 0.0190) but not for women or overall. However, there was an increase in IS hospitalizations in men 35–44 years of age (+37.5%; p = 0.0019). The length of stay (LOS) of IS patients declined in nearly all subgroups (overall -20.8%, p<0.0001) whereas no change in LOS was observed for patients with ICH or SAH. In-hospital mortality decreased in patients with IS (-42.8%; p = 0.0092) but remained unchanged in patients with ICH or SAH. Conclusions Stroke hospitalizations of young people declined in Finland, except for men 35–44 years of age for whom IS hospitalizations increased. Declining LOS and in-hospital mortality of IS patients suggests admission of less severe cases, improved care or both.