Atrial Fibrillation Risk Assessment after Embolic Stroke of Undetermined Source.

OBJECTIVE: Approximately 20% of strokes are embolic strokes of undetermined source (ESUS). Undetected atrial fibrillation (AF) remains an important cause. Yet, oral anticoagulation in unselected ESUS patients failed in secondary stroke prevention. Guidance on effective AF detection is lacking. Here, we introduce a novel, non-invasive AF risk assessment after ESUS. METHODS: Catch-Up ESUS is an investigator-initiated, observational cohort study conducted between 2018 and 2019 at the Munich University Hospital. Besides clinical characteristics, patients received ≥72 h digital electrocardiogram recordings to generate the rhythm irregularity burden. Uni- and multivariable regression models predicted the primary endpoint of incident AF, ascertained by standardized follow-up including implantable cardiac monitors. Predictors included the novel rhythm irregularity burden constructed from digital electrocardiogram recordings. We independently validated our model in ESUS patients from the University Hospital Tübingen, Germany. RESULTS: A total of 297 ESUS patients were followed for 15.6 ± 7.6 months. Incident AF (46 patients, 15.4%) occurred after a median of 105 days (25th to 75th percentile 31-33 days). Secondary outcomes were recurrent stroke in 7.7% and death in 6.1%. Multivariable-adjusted analyses identified the rhythm irregularity burden as the strongest AF-predictor (hazard ratio 3.12, 95% confidence interval 1.62-5.80, p < 0001) while accounting for the known risk factors age, CHA2 DS2 -VASc-Score, and NT-proBNP. Independent validation confirmed the rhythm irregularity burden as the most significant AF-predictor (hazard ratio 2.20, 95% confidence interval 1.45-3.33, p < 0001). INTERPRETATION: The novel, non-invasive, electrocardiogram-based rhythm irregularity burden may help adjudicating AF risk after ESUS, and subsequently guide AF-detection after ESUS. Clinical trials need to clarify if high-AF risk patients benefit from tailored secondary stroke prevention. ANN NEUROL 2023;93:479-488.

[Ethical Attitudes and Handling in Prenatal Conflict Situations - A Survey among Obstetricians and Prenatal Diagnosticians in Germany]. / Ethische Haltungen und Umgang in pränatalen Konfliktsituationen ­ eine Umfrage unter Geburtshelfern und Pränataldiagnostikern in Deutschland.

OBJECTIVE: Various studies have shown that belonging to a professional group has an influence on ethical attitudes. The aim of this study was to assess and compare the attitudes and approaches of obstetrical specialists and prenatal diagnosticians in prenatal conflict situations. METHODS: Explorative cross-sectional online survey among tertiary perinatal care centers and prenatal diagnosticians with DEGUM Level II/III in Germany. The questionnaire included questions on ethical attitudes in the perinatal context and a case presentation of a fetal hypoplastic left heart syndrome. RESULTS: The response rate was 57.1% (310/543). 55.5% of the respondents practiced both obstetrics and prenatal diagnostics, 24.5% exclusively prenatal diagnostics, and 14.2% purely obstetrics. 27% agreed with the statement "An uncertain prognosis justifies pregnancy termination". For complex fetal malformations joint interdisciplinary counseling was advocated by 98.3%. Addressing the option of postnatal palliative treatment in a case of a hypoplastic left heart syndrome was accepted by 84.3% across all professional groups, while mentioning fetocide was more frequently cited as an option by prenatal diagnosticians than by obstetricians (57.7% vs. 34.1%). CONCLUSION: Interdisciplinary prenatal parental counseling in complex fetal malformations is uniformly advocated by prenatal diagnosticians and obstetricians in Germany. However, different ethical attitudes appear among specialists groups with regard to the option of termination of pregnancy.

German obstetrician's self-reported attitudes and handling in threatening preterm birth at the limits of viability.

OBJECTIVES: Antenatal treatment and information influences the course of pregnancy and parental decision-making in cases of threatened prematurity on the borderline of viability. Numerous studies have shown significant interprofessional differences in assessing ethical boundary decisions; hence, this study aimed to evaluate obstetricians attitudes, practices and antenatal parental counseling regarding threatened preterm birth in Germany. METHODS: An anonymous online questionnaire was administered to 543 obstetricians at tertiary perinatal centers and prenatal diagnostic centers in Germany. The survey contained questions on basic ethical issues assessed using the Likert scale and a case vignette regarding the practical procedures of an imminent extreme premature birth at 23 1/7 gestational weeks. RESULTS: In the case of unstoppable preterm birth, 15 % of clinicians said they would carry out a cesarean section; however, specialists from centers with a high number of very low birth weight infants would do so significantly more often. Among respondents, 29.8 % did not take any therapeutic measures without discussing the child's treatment options with their parents, 19.9 % refused to offer actionable advice to the parents, and 57 % said they would advise parents to seek intensive care treatment for the child with the option of changing treatment destination in the event of serious complications. Moreover, 84 % said they would provide information together with neonatologists. CONCLUSIONS: Joint counseling with neonatologists is widely accepted. The size of the perinatal center significantly influences the practical approach to threatened preterm births. Respect for parents' decision-making autonomy regarding the child's treatment options is central and influences therapy initiation.

Structured magnetic resonance imaging-based characterization of the marginal vein reveals limits of the Weber-classification.

Background: The marginal vein (MV) is a persisting embryonic vein located at the lateral aspect of the lower limb. The Weber-classification, which was developed on the basis of phlebography in the 1990s, is the only existing classification system for this rare disease. Aim of this study was the structured characterization of the lateral marginal vein (MV) using magnetic resonance imaging (MRI) and evaluation of the applicability of the Weber-classification. Patients and methods: Institutional Review Board approval was obtained for this retrospective, single-center study. All patients who underwent contrast-enhanced MRI (using a prospectively determined protocol) of the untreated MV were included. MV anatomy and associated findings were characterized in a structured way taking into account the criteria of the Weber-classification for MV: inflow, outflow and extension. If three criteria of the Weber-classification were fulfilled the MV was categorized as "classifiable according to Weber". The MV was categorized as "partially classifiable according to Weber", if two criteria were met and as "not classifiable according to Weber" if less than two criteria were applicable. Results: 56 imaging studies of 58 MV (7 thoracoabdominal, 51 lower extremities) were reviewed. 18/51 MV of the lower extremities were "classifiable" according to the Weber-classification. 33/51 lower extremity MV were not definitely categorized according to the Weber-classification: 19/51 MV were "partially classifiable" and 14/51 MV were "not classifiable". 30/51 MV presented with hypoplastic, 1/51 with aplastic deep venous system. 34/51 lower extremity and 6/7 thoracoabdominal MV were associated with an additional vascular malformation (VM). Conclusions: MRI is suitable for detailed anatomic characterization of the MV and reveals additional therapy relevant findings like associated VM. The Weber-classification was not applicable in most cases, reflecting its limits and the heterogeneity of this rare disease. Structured reports rather than an obsolete classification system should be preferred for MRI of the MV.

Bright on the right feels right: SQUARC compatibility is hedonically marked.

According to the Spatial Quantity Association of Response Codes (SQUARC), people hold a mental association between horizontal position and quantity (lower quantities left, higher quantities right). While a large body of research has explored this effect for response speed and judgment accuracy, the affective downstream consequences of the SQUARC remain unexplored. Aiming to address this gap, the present two experiments (pre-registered, total N = 521) investigated whether stimulus arrangements that are compatible with the SQUARC for luminance are affectively preferred to stimulus arrangements that are incompatible. SQUARC-compatible square arrangements (dark-left, bright-right) were preferred over SQUARC-incompatible square arrangements (dark-right, bright-left). The preference for SQUARC compatibility was not moderated by the horizontal orientation of the response scale. Our results confirm the direction of the spatial-luminance association and provide initial support that the cognitive processing of SQUARC compatibility is hedonically marked and appears sufficient to impact affective evaluations.

KCTD12 Auxiliary Proteins Modulate Kinetics of GABAB Receptor-Mediated Inhibition in Cholecystokinin-Containing Interneurons.

Cholecystokinin-expressing interneurons (CCK-INs) mediate behavior state-dependent inhibition in cortical circuits and themselves receive strong GABAergic input. However, it remains unclear to what extent GABAB receptors (GABABRs) contribute to their inhibitory control. Using immunoelectron microscopy, we found that CCK-INs in the rat hippocampus possessed high levels of dendritic GABABRs and KCTD12 auxiliary proteins, whereas postsynaptic effector Kir3 channels were present at lower levels. Consistently, whole-cell recordings revealed slow GABABR-mediated inhibitory postsynaptic currents (IPSCs) in most CCK-INs. In spite of the higher surface density of GABABRs in CCK-INs than in CA1 principal cells, the amplitudes of IPSCs were comparable, suggesting that the expression of Kir3 channels is the limiting factor for the GABABR currents in these INs. Morphological analysis showed that CCK-INs were diverse, comprising perisomatic-targeting basket cells (BCs), as well as dendrite-targeting (DT) interneurons, including a previously undescribed DT type. GABABR-mediated IPSCs in CCK-INs were large in BCs, but small in DT subtypes. In response to prolonged activation, GABABR-mediated currents displayed strong desensitization, which was absent in KCTD12-deficient mice. This study highlights that GABABRs differentially control CCK-IN subtypes, and the kinetics and desensitization of GABABR-mediated currents are modulated by KCTD12 proteins.

The CCAS-scale in hereditary ataxias: helpful on the group level, particularly in SCA3, but limited in individual patients.

BACKGROUND: A brief bedside test has recently been introduced by Hoche et al. (Brain, 2018) to screen for the Cerebellar Cognitive Affective Syndrome (CCAS) in patients with cerebellar disease. OBJECTIVE: This multicenter study tested the ability of the CCAS-Scale to diagnose CCAS in individual patients with common forms of hereditary ataxia. METHODS: A German version of the CCAS-Scale was applied in 30 SCA3, 14 SCA6 and 20 FRDA patients, and 64 healthy participants matched for age, sex, and level of education. Based on original cut-off values, the number of failed test items was assessed, and CCAS was considered possible (one failed item), probable (two failed items) or definite (three failed items). In addition a total sum raw score was calculated. RESULTS: On a group level, failed items were significantly higher and total sum scores were significantly lower in SCA3 patients compared to matched controls. SCA6 and FRDA patients performed numerically below controls, but respective group differences failed to reach significance. The ability of the CCAS-Scale to diagnose CCAS in individual patients was limited to severe cases failing three or more items. Milder cases failing one or two items showed a great overlap with the performance of controls exhibiting a substantial number of false-positive test results. The word fluency test items differentiated best between patients and controls. CONCLUSIONS: As a group, SCA3 patients performed below the level of SCA6 and FRDA patients, possibly reflecting additional cerebral involvement. Moreover, the application of the CCAS-Scale in its present form results in a high number of false-positive test results, that is identifying controls as patients, reducing its usefulness as a screening tool for CCAS in individual patients.

The Wlds transgene reduces axon loss in a Charcot-Marie-Tooth disease 1A rat model and nicotinamide delays post-traumatic axonal degeneration.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy and a duplication of the peripheral myelin protein of 22 kDa (PMP22) gene causes the most frequent subform CMT1A. Clinical impairments are determined by the amount of axonal loss. Axons of the spontaneous mouse mutant Wallerian degeneration slow (Wlds) show markedly reduced degeneration following various types of injuries. Protection is conferred by a chimeric Wlds gene encoding an N-terminal part of ubiquitination factor Ube4b and full length nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1). Nmnat1 enzyme generates nicotinamide adenine dinucleotide (NAD) from nicotinamide mononucleotide. Here, in a Pmp22 transgenic animal model of Charcot-Marie-Tooth disease type 1A (CMT rat), the Wlds transgene reduced axonal loss and clinical impairments without altering demyelination. Furthermore, nicotinamide - substrate precursor of the Nmnat1 enzyme - transiently delayed posttraumatic axonal degeneration in an in vivo model of acute peripheral nerve injury, but to a lower extent than Wlds. In contrast, 8 weeks of nicotinamide treatment did not influence axonal loss or clinical manifestations in the CMT rat. Therefore, nicotinamide can partially substitute for the protective Wlds effect in acute traumatic, but not in chronic secondary axonal injury. Future studies are needed to develop axon protective therapy in CMT1A which may be combined with therapeutic strategies aimed at downregulation of toxic PMP22 overexpression.

Manganese superoxide dismutase and aldehyde dehydrogenase deficiency increase mitochondrial oxidative stress and aggravate age-dependent vascular dysfunction.

AIMS: Imbalance between pro- and antioxidant species (e.g. during aging) plays a crucial role for vascular function and is associated with oxidative gene regulation and modification. Vascular aging is associated with progressive deterioration of vascular homeostasis leading to reduced relaxation, hypertrophy, and a higher risk of thrombotic events. These effects can be explained by a reduction in free bioavailable nitric oxide that is inactivated by an age-dependent increase in superoxide formation. In the present study, mitochondria as a source of reactive oxygen species (ROS) and the contribution of manganese superoxide dismutase (MnSOD, SOD-2) and aldehyde dehydrogenase (ALDH-2) were investigated. METHODS AND RESULTS: Age-dependent effects on vascular function were determined in aortas of C57/Bl6 wild-type (WT), ALDH-2(-/-), MnSOD(+/+), and MnSOD(+/-) mice by isometric tension measurements in organ chambers. Mitochondrial ROS formation was measured by luminol (L-012)-enhanced chemiluminescence and 2-hydroxyethidium formation with an HPLC-based assay in isolated heart mitochondria. ROS-mediated mitochondrial DNA (mtDNA) damage was detected by a novel and modified version of the fluorescent-detection alkaline DNA unwinding (FADU) assay. Endothelial dysfunction was observed in aged C57/Bl6 WT mice in parallel to increased mitochondrial ROS formation and oxidative mtDNA damage. In contrast, middle-aged ALDH-2(-/-) mice showed a marked vascular dysfunction that was similar in old ALDH-2(-/-) mice suggesting that ALDH-2 exerts age-dependent vasoprotective effects. Aged MnSOD(+/-) mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage. CONCLUSION: The correlation between mtROS formation and acetylcholine-dependent relaxation revealed that mitochondrial radical formation significantly contributes to age-dependent endothelial dysfunction.

AT1-receptor blockade by telmisartan upregulates GTP-cyclohydrolase I and protects eNOS in diabetic rats.

Several enzymatic sources of reactive oxygen species (ROS) were described as potential reasons of eNOS uncoupling in diabetes mellitus. In the present study, we investigated the effects of AT1-receptor blockade with chronic telmisartan (25 mg/kg/day, 6.5 weeks) therapy on expression of the BH4-synthesizing enzyme GTP-cyclohydrolase I (GCH-I), eNOS uncoupling, and endothelial dysfunction in streptozotocin (STZ, 60 mg/kg iv, 7 weeks)-induced diabetes mellitus (type I). Telmisartan therapy did not modify blood glucose and body weight. Aortas from diabetic animals had vascular dysfunction as revealed by isometric tension studies (acetylcholine and nitroglycerin potency). Vascular and cardiac ROS produced by NADPH oxidase, mitochondria, eNOS, and xanthine oxidase were increased in the diabetic group as was the expression of NADPH oxidase subunits at the protein level. The expression of GCH-I and the phosphorylation of eNOS at Ser1177 was decreased by STZ treatment. Therapy with telmisartan normalized these parameters. The present study demonstrates for the first time that AT1-receptor blockade by telmisartan prevents downregulation of the BH4 synthase GCH-I and thereby eNOS uncoupling in experimental diabetes. In addition, telmisartan inhibits activation of superoxide sources like NADPH oxidase, mitochondria, and xanthine oxidase. These effects may explain the beneficial effects of telmisartan on endothelial dysfunction in diabetes.

ALDH-2 deficiency increases cardiovascular oxidative stress--evidence for indirect antioxidative properties.

Mitochondrial aldehyde dehydrogenase (ALDH-2) reduces reactive oxygen species (ROS) formation related to toxic aldehydes; additionally, it provides a bioactivating pathway for nitroglycerin. Since acetaldehyde, nitroglycerin, and doxorubicin treatment provoke mitochondrial oxidative stress, we used ALDH-2(-/-) mice and purified recombinant human ALDH-2 to test the hypothesis that ALDH-2 has an indirect antioxidant function in mitochondria. Antioxidant capacity of purified ALDH-2 was comparable to equimolar doses of glutathione, cysteine, and dithiothreitol; mitochondrial oxidative stress was comparable in C57Bl6 and ALDH-2(-/-) mice after acute challenges with nitroglycerin or doxorubicin, whereas chronic acetaldehyde, nitroglycerin, and doxorubicin treatment dose-dependently increased mitochondrial ROS formation and impaired endothelial function to a greater extent in ALDH-2(-/-) mice. Maximal nitroglycerin dose applied in vivo lead to a "super-desensitized" nitroglycerin response in isolated ALDH-2(-/-) aortas, inaccessible in C57Bl6 mice. Our results suggest that ALDH-2 has an indirect antioxidative property independent of its thiol-moiety in disease states of cardiovascular oxidative stress.

Drug specificity and intestinal membrane localization of human organic cation transporters (OCT).

This study was performed to investigate which human organic cation transporter, hOCT1, hOCT2 or hOCT3, participates with regard to cation specificity and membrane localization in the intestinal absorption of orally available cationic drugs. Inhibition of N-[methyl-3H]4-phenylpyridinium ([3H]MPP+) uptake by various compounds into Caco-2 cells and into cells (HEK-293 or CHO) that were stably transfected with hOCT1, hOCT2 or hOCT3 was compared. The uptake of [3H]MPP+ into Caco-2 cells was inhibited by atropine, butylscopolamine, clonidine, diphenhydramine, etilefrine, quinine and ranitidine with IC50 values between 6 microM and 4 mM. Transepithelial, apical to basal flux of [3H]MPP+ across Caco-2 cell monolayers was also strongly inhibited by these compounds. The inhibitory potency of the cationic drugs and prototypical organic cations at Caco-2 cells correlated well with the inhibitory potency measured at CHO-hOCT3 cells but much less with that at HEK-hOCT1 and -hOCT2 cells. This is functional evidence for the predominant role of hOCT3. Etilefrine and atropine were specifically transported into CHO cells by hOCT3. In Caco-2 cells, the mRNA of all three hOCT and the proteins hOCT2 and hOCT3 were detected. More importantly, immunocytochemical analyses of human jejunum revealed for the first time that hOCT3 is localized to the brush border membrane whereas hOCT1 immunolabeling was mainly observed at the lateral membranes of the enterocytes.

Quinpramine ameliorates rat experimental autoimmune neuritis and redistributes MHC class II molecules.

Activation of inflammatory cells is central to the pathogenesis of autoimmune demyelinating diseases of the peripheral nervous system. The novel chimeric compound quinpramine--generated from imipramine and quinacrine--redistributes cholesterol rich membrane domains to intracellular compartments. We studied the immunological and clinical effects of quinpramine in myelin homogenate induced Lewis rat experimental autoimmune neuritis (EAN), a model system for acute human inflammatory neuropathies, such as the Guillain-Barré syndrome. EAN animals develop paresis of all limbs due to autoimmune inflammation of peripheral nerves. Quinpramine treatment ameliorated clinical disease severity of EAN and infiltration of macrophages into peripheral nerves. It reduced expression of MHC class II molecules on antigen presenting cells and antigen specific T cell proliferation both in vitro and in vivo. Quinpramine exerted its anti-proliferatory effect on antigen presenting cells, but not on responder T cells. Our data suggest that quinpramine represents a candidate pharmaceutical for inflammatory neuropathies.

Chemical model systems for cellular nitros(yl)ation reactions.

S-nitros(yl)ation belongs to the redox-based posttranslational modifications of proteins but the underlying chemistry is controversial. In contrast to current concepts involving the autoxidation of nitric oxide ((.)NO, nitrogen monoxide), we and others have proposed the formation of peroxynitrite (oxoperoxonitrate (1(-))as an essential intermediate. This requires low cellular fluxes of (.)NO and superoxide (UO2(-)), for which model systems have been introduced. We here propose two new systems for nitros(yl)ation that avoid the shortcomings of previous models. Based on the thermal decomposition of 3-morpholinosydnonimine,equal fluxes of (.)NO and UO2(-) were generated and modulated by the addition of (.)NO donors or Cu,Zn superoxide dismutase. As reactants for S-nitros(yl)ation, NADP+-dependent isocitrate dehydrogenase and glutathione were employed, for which optimal S-nitros(yl)ation was observed at nanomolar fluxes of (.)NO and UO2(-) at a ratio of about 3:1. The previously used reactants phenol and diaminonaphthalene (C- and Nnitrosation)demonstrated potential participation of multiple pathways for nitros(yl)ation. According to our data, neither peroxynitrite nor autoxidation of UNO was as efficient as the 3 (.)NO/1 UO2(-) system in mediating S-nitros(yl)ation. In theory this could lead to an elusive nitrosonium (nitrosyl cation)-like species in the first step and to N2O3 in the subsequent reaction. Which of these two species or whether both together will participate in biological S-nitros(yl)ation remains to be elucidated. Finally, we developed several hypothetical scenarios to which the described (.)NO/UO2-flux model could apply, providing conditions that allow either direct electrophilic substitution at a thiolate or S-nitros(yl)ation via transnitrosation from S-nitrosoglutathione.

First evidence for a crosstalk between mitochondrial and NADPH oxidase-derived reactive oxygen species in nitroglycerin-triggered vascular dysfunction.

Chronic nitroglycerin treatment results in development of nitrate tolerance associated with endothelial dysfunction (ED). We sought to clarify how mitochondria- and NADPH oxidase (Nox)-derived reactive oxygen species (ROS) contribute to nitrate tolerance and nitroglycerin-induced ED. Nitrate tolerance was induced by nitroglycerin infusion in male Wistar rats (100 microg/h/4 day) and in C57/Bl6, p47(phox/) and gp91(phox/) mice (50 microg/h/4 day). Protein and mRNA expression of Nox subunits were unaltered by chronic nitroglycerin treatment. Oxidative stress was determined in vascular rings and mitochondrial fractions of nitroglycerin-treated animals by L-012 enhanced chemiluminescence, revealing a dominant role of mitochondria for nitrate tolerance development. Isometric tension studies revealed that genetic deletion or inhibition (apocynin, 0.35 mg/h/4 day) of Nox improved ED, whereas nitrate tolerance was unaltered. Vice versa, nitrate tolerance was attenuated by co-treatment with the respiratory chain complex I inhibitor rotenone (100 microg/h/4 day) or the mitochondrial permeability transition pore blocker cyclosporine A (50 microg/h/4 day). Both compounds improved ED, suggesting a link between mitochondrial and Nox-derived ROS. Mitochondrial respiratory chain-derived ROS are critical for the development of nitrate tolerance, whereas Nox-derived ROS mediate nitrate tolerance-associated ED. This suggests a crosstalk between mitochondrial and Nox-derived ROS with distinct mechanistic effects and sites for pharmacological intervention.

Transport of clonidine at cultured epithelial cells (JEG-3) of the human placenta.

PURPOSE: This study was performed to characterize the uptake of the antihypertensive drug clonidine at epithelial cells of the human placenta (JEG-3) and its interaction with other cationic drugs. METHODS: Uptake of [3H]clonidine into cells of the human choriocarcinoma cell line JEG-3 was investigated. RESULTS: Uptake of [3H]clonidine into JEG-3 cells reached its maximum on the eighth day after seeding. Uptake of [3H]clonidine was linear for up to 1 min, Na+ independent, but strongly H+ dependent. The uptake rate of clonidine was saturable with an affinity constant (Kt) of 1.1 mM and a Vmax value of 27 nmol x min(-1) x mg(-1) of protein. Several cationic drugs such as clonidine, quinine, quinidine, and diphenhydramine strongly inhibited the [3H]clonidine uptake with Ki values around 1 mM. CONCLUSIONS: Clonidine is transported across the placental epithelium by a carrier mediated process.