Insulin resistance in mice lacking neuronal nitric oxide synthase is related to an alpha-adrenergic mechanism

BACKGROUND: nitric oxide (NO) plays an important role in the regulation of cardiovascular and glucose homeostasis. Mice lacking the gene encoding the neuronal isoform of nitric oxide synthase (nNOS) are insulin-resistant, but the underlying mechanism is unknown. nNOS is expressed in skeletal muscle tissue where it may regulate glucose uptake. Alternatively, nNOS driven NO synthesis may facilitate skeletal muscle perfusion and substrate delivery. Finally, nNOS dependent NO in the central nervous system may facilitate glucose disposal by decreasing sympathetic nerve activity. METHODS: in nNOS null and control mice, we studied whole body glucose uptake and skeletal muscle blood flow during hyperinsulinaemic clamp studies in vivo and glucose uptake in skeletal muscle preparations in vitro. We also examined the effects of alpha-adrenergic blockade (phentolamine) on glucose uptake during the clamp studies. RESULTS: as expected, the glucose infusion rate during clamping was roughly 15 percent lower in nNOS null than in control mice (89 (17) vs 101 (12) [-22 to -2]). Insulin stimulation of muscle blood flow in vivo, and intrinsic muscle glucose uptake in vitro, were comparable in the two groups. Phentolamine, which had no effect in the wild-type mice, normalised the insulin sensitivity in the mice lacking the nNOS gene. CONCLUSIONS: insulin resistance in nNOS null mice was not related to defective insulin stimulation of skeletal muscle perfusion and substrate delivery or insulin signaling in the skeletal muscle cell, but to a sympathetic alpha-adrenergic mechanism.

Role of glial cells in the functional expression of LL-37/rat cathelin-related antimicrobial peptide in meningitis

Antimicrobial peptides are intrinsic to the innate immune system in many organ systems, but little is known about their expression in the central nervous system. We examined cerebrospinal fluid (CSF) and serum from patients with active bacterial meningitis to assess antimicrobial peptides and possible bactericidal properties of the CSF. We found antimicrobial peptides (human cathelicidin LL-37) in the CSF of patients with bacterial meningitis but not in control CSF. We next characterized the expression, secretion, and bactericidal properties of rat cathelin-related antimicrobial peptide, the homologue of the human LL-37, in rat astrocytes and microglia after incubation with different bacterial components. Using real-time polymerase chain reaction and Western blotting, we determined that supernatants from both astrocytes and microglia incubated with bacterial component supernatants had antimicrobial activity. The expression of rat cathelin-related antimicrobial peptide in rat glial cells involved different signal transduction pathways and was induced by the inflammatory cytokines interleukin 1beta and tumor necrosis factor. In an experimental model of meningitis, infant rats were intracisternally infected with Streptococcus pneumoniae, and rat cathelin-related antimicrobial peptide was localized in glia, choroid plexus, and ependymal cells by immunohistochemistry. Together, these results suggest that cathelicidins produced by glia and other cells play an important part in the innate immune response against pathogens in central nervous system bacterial infections.

Functional regeneration of intraspinal connections in a new in vitro model

Abstract—Regeneration in the adult mammalian spinal cord is limited due to intrinsic properties of mature neurons and a hostile environment, mainly provided by central nervous system myelin and reactive astrocytes. Recent results indicate that propriospinal connections are a promising target for intervention to improve functional recovery. To study this functional regeneration in vitro we developed a model consisting of two organotypic spinal cord slices placed adjacently on multi-electrode arrays. The electrodes allow us to record the spontaneously occurring neuronal activity, which is often organized in network bursts. Within a few days in vitro (DIV), these bursts become synchronized between the two slices due to the formation of axonal connections. We cut them with a scalpel at different time points in vitro and record the neuronal activity 3 weeks later. The functional recovery ability was assessed by calculating the percentage of synchronized bursts between the two slices. We found that cultures lesioned at a young age (7–9 DIV) retained the high regeneration ability of embryonic tissue. However, cultures lesioned at older ages (>19 DIV) displayed a distinct reduction of synchronized activity. This reduction was not accompanied by an inability for axons to cross the lesion site. We show that functional regeneration in these old cultures can be improved by increasing the intracellular cAMP level with Rolipram or by placing a young slice next to an old one directly after the lesion. We conclude that co-cultures of two spinal cord slices are an appropriate model to study functional regeneration of intraspinal connections.

Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes

Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.

Genome-wide association study on dimethylarginines reveals novel AGXT2 variants associated with heart rate variability but not with overall mortality.

AIMS The purpose of this study was to identify novel genetic variants influencing circulating asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) levels and to evaluate whether they have a prognostic value on cardiovascular mortality. METHODS AND RESULTS We conducted a genome-wide association study on the methylarginine traits and investigated the predictive value of the new discovered variants on mortality. Our meta-analyses replicated the previously known locus for ADMA levels in DDAH1 (rs997251; P = 1.4 × 10(-40)), identified two non-synomyous polymorphisms for SDMA levels in AGXT2 (rs37369; P = 1.4 × 10(-40) and rs16899974; P = 1.5 × 10(-38)) and one in SLC25A45 (rs34400381; P = 2.5 × 10(-10)). We also fine-mapped the AGXT2 locus for further independent association signals. The two non-synonymous AGXT2 variants independently associated with SDMA levels were also significantly related with short-term heart rate variability (HRV) indices in young adults. The major allele (C) of the novel non-synonymous rs16899974 (V498L) variant associated with decreased SDMA levels and an increase in the ratio between the low- and high-frequency spectral components of HRV (P = 0.00047). Furthermore, the SDMA decreasing allele (G) of the non-synomyous SLC25A45 (R285C) variant was associated with a lower resting mean heart rate during the HRV measurements (P = 0.0046), but not with the HRV indices. None of the studied genome-wide significant variants had any major effect on cardiovascular or total mortality in patients referred for coronary angiography. CONCLUSIONS AGXT2 has an important role in SDMA metabolism in humans. AGXT2 may additionally have an unanticipated role in the autonomic nervous system regulation of cardiac function.

Infection of organotypic slice cultures from rat central nervous tissue with Neospora caninum: an alternative approach to study host-parasite interactions

Neospora caninum is an apicomplexan parasite which has emerged as an important cause of bovine abortion worldwide. Abortion is usually triggered by reactivation of dormant bradyzoites during pregnancy and subsequent congenital infection of the foetus, where the central nervous system appears to be most frequently affected. We here report on an organotypic tissue culture model for Neospora infection which can be used to study certain aspects of the cerebral phase of neosporosis within the context of a three-dimensionally organised neuronal network. Organotypic slice cultures of rat cortical tissue were infected with N. caninum tachyzoites, and the kinetics of parasite proliferation, as well as the proliferation-inhibitory effect of interferon-gamma (IFN-gamma), were monitored by either immunofluorescence, transmission electron microscopy, and a quantitative PCR-assay using the LightCycler instrument, respectively. In addition, the neuronal cytoskeletal elements, namely glial acidic protein filaments as well as actin microfilament bundles were shown to be largely colocalising with the pseudocyst periphery. This organotypic culture model for cerebral neosporosis provides a system, which is useful to study the proliferation, ultrastructural characteristics, development, and the interactions of N. caninum within the context of neuronal tissue, which at the same time can be modulated and influenced under controlled conditions, and will be useful in the future to gain more information on the cerebral phase of neosporosis.

Lazy lips: hyperkalemia and acute tetraparesis-a case report from an urban emergency department.

A 58-year-old male patient was admitted to our emergency department at a large university hospital due to acute onset of general weakness. It was reported that the patient was bradycardic at 30/min and felt an increasing weakness of the limbs. At admission to the emergency department, the patient was not feeling any discomfort and denied dyspnoea or pain. The primary examination of the nervous system showed the cerebral nerves II-XII intact, muscle strength of the lower extremities was 4/5, and a minimal sensory loss of the left hemisphere was found. In addition, the patient complained about lazy lips. During ongoing examinations, the patient developed again symptomatic bradycardia, accompanied by complete tetraplegia. The following blood test showed severe hyperkalemia probably induced by use of aldosterone antagonists as the cause of the patient's neurologic symptoms. Hyperkalemia is a rare but treatable cause of acute paralysis that requires immediate treatment. Late diagnosis can delay appropriate treatment leading to cardiac arrhythmias and arrest.

[Anesthesia management in implantation of baroreceptor stimulators].

Baroreceptor stimulators are novel implantable devices that activate the carotid baroreceptor reflex. This results in a decrease in activity of the sympathetic nervous system and inhibition of the renin-angiotensin-aldosterone system. In patients with drug-resistant hypertension, permanent electrical activation of the baroreceptor reflex results in blood pressure reduction and cardiac remodeling. For correct intraoperative electrode placement at the carotid bifurcation, the baroreceptor reflex needs to be activated several times. Many common anesthetic agents, such as inhalation anesthetics and propofol dampen or inhibit the baroreceptor reflex and complicate or even prevent successful placement. Therefore, a specific anesthesia and pharmacological management is necessary to ensure successful implantation of baroreceptor reflex stimulators.

Das Elektrokardiogramm des Sporttreibenden und der Sport-assoziierte plötzliche Herztod.

Regelmässiges körperliches Training induziert strukturelle, elektrische und funktionelle Anpassungen des Herzens. Die grösste Herausforderung für den Arzt liegt darin, Veränderungen hinweisend für eine strukturelle Herzerkrankung von physiologischen, trainingsassoziierten Anpassungen im Sinne eines 'Athlete's heart' zu unterscheiden. Bei zugrundliegender Kardiopathie ist sportliche Aktivität nicht die Ursache, sondern kann ein Trigger für belastungsabhängige Tachyarrhythmien bzw. für den belastungsabhängigen plötzlichen Herztod (SCD) sein. Um Athleten mit einer kardialen Grunderkrankung und erhöhtem Risiko für einen SCD frühzeitig zu identifizieren wird in Europa ein Preparticipation Screening empfohlen, welches von der Schweizerischen Gesellschaft für Sportmedizin (SGSM) übernommen wurde. Dieses Screening umfasst neben der spezifischen Anamnese und der Herzauskultation auch ein Ruhe-Elektrokardiogramm (Ruhe-EKG). Aufgrund der hohen Anzahl falsch-positiver EKG-Befunde wurden in den letzten Jahren die Beurteilungskriterien des Athleten-EKGs wiederholt angepasst, die Sensitivität und insbesondere auch die Spezifität konnte mit den „verfeinerten Seattle Kriterien“ 2014 deutlich verbessert werden. Der frühen Repolarisation galt in den letzten Jahren ein Hauptaugenmerk: neben dem (Ausdauer-) Training besteht eine klare Assoziation zum männlichen Geschlecht, zur Ethnie, zu den Veränderungen des vegetativen Nervensystems und zu erhöhten QRS-Voltage-Kriterien.

Sex differences in heart rate variability: a longitudinal study in international elite cross-country skiers

PURPOSE: Exercise-related sudden cardiac deaths (SCD) occur with a striking male predominance. A higher sympathetic tone in men has been suggested as risk factor for SCD. Elite athletes have the highest risk for exercise-related SCD. We aimed to analyze the autonomic nervous system of elite cross-country skiers from Norway, Russia and Switzerland in supine position and after orthostatic challenge in various training periods (TP). METHOD: Measurements of heart rate variability (HRV) were performed on a weekly basis over 1 year using an orthostatic challenge test with controlled breathing. Main outcome parameters were the high-frequency power in supine position (HFsupine) as marker of cardiac parasympathetic activity and the low-frequency/high-frequency power ratio after orthostatic challenge (LF/HFstand) as marker of cardiac sympathetic activation. Training intensity and duration were recorded daily and expressed as training strain. The training year was divided into three TPs. An average of weekly HRV measurements was calculated for each TP. RESULT: Female (n = 19, VO2max 62.0 +/- 4.6 ml kg(-1) min(-1), age 25.8 +/- 4.3 years) and male (n = 16, VO2max 74.3 +/- 6.3 ml kg(-1) min(-1), age 24.4 +/- 4.2 years) athletes were included. Training strain was comparable between sexes (all p > 0.05) and changed between TPs (all p < 0.05) while no HRV parameters changed over time. There were no sex differences in HFsupine while the LF/HFstand was significantly higher in male athletes in all TPs. CONCLUSION: For a comparable amount of training, male athletes showed constantly higher markers of sympathetic activity after a provocation maneuver. This may explain part of the male predominance in sports-related SCD.

The Gut Microbiome and Cirrhosis: Basic Aspects

In this chapter the basic aspects helping to understand the microbiome in terms of quantity, diversity, complexity, function, and interaction with the host are discussed. First the nomenclature, definitions of taxa, and measures of diversity as well as methods to unravel this kingdom are outlined. A brief summary on its physiological relevance for general health and the functions exerted specifically by the microbiome is presented. Differences in the composition of the microbiome along the gastrointestinal tract and across the gut wall and its interindividual variations, enterotypes, and stability are highlighted. The reader will be familiarized with all different modulators impacting on the microbiome, namely, intrinsic and extrinsic factors. Intrinsic factors include gastrointestinal secretions (gastric acid, bile, pancreatic juice, mucus), antimicrobial peptides, motility, enteric nervous system, and host genotype. Extrinsic factors are mainly dietary choices, hygiene, stress, alcohol consumption, exercise, and medications. The second part of the chapter focuses on quantitative and qualitative changes in microbiome in liver cirrhosis. The mechanisms contributing to dysbiosis, small intestinal bacterial overgrowth, and bacterial translocation are delineated underscoring their role for the liver-gut axis.

Inherited progressive cardiac conduction disorders.

PURPOSE OF REVIEW Progressive cardiac conduction disorder (PCCD) is an inherited cardiac disease that may present as a primary electrical disease or be associated with structural heart disease. In this brief review, we present recent clinical, genetic, and molecular findings relating to PCCD. RECENT FINDINGS Inherited PCCD in structurally normal hearts has been found to be linked to genetic variants in the ion channel genes SCN5A, SCN1B, SCN10A, TRPM4, and KCNK17, as well as in genes coding for cardiac connexin proteins. In addition, several SCN5A mutations lead to 'cardiac sodium channelopathy overlap syndrome'. Other genes coding for cardiac transcription factors, such as NKX2.5 and TBX5, are involved in the development of the cardiac conduction system and in the morphogenesis of the heart. Mutations in these two genes have been shown to cause cardiac conduction disorders associated with various congenital heart defects. SUMMARY PCCD is a hereditary syndrome, and genetic variants in multiple genes have been described to date. Genetic screening and identification of the causal mutation are crucial for risk stratification and family counselling.

The Ets domain transcription factor Erm distinguishes rat satellite glia from Schwann cells and is regulated in satellite cells by neuregulin signaling

Distinct glial cell types of the vertebrate peripheral nervous system (PNS) are derived from the neural crest. Here we show that the expression of the Ets domain transcription factor Erm distinguishes satellite glia from Schwann cells beginning early in rat PNS development. In developing dorsal root ganglia (DRG), Erm is present both in presumptive satellite glia and in neurons. In contrast, Erm is not detectable at any developmental stage in Schwann cells in peripheral nerves. In addition, Erm is downregulated in DRG-derived glia adopting Schwann cell traits in culture. Thus, Erm is the first described transcription factor expressed in satellite glia but not in Schwann cells. In culture, the Neuregulin1 (NRG1) isoform GGF2 maintains Erm expression in presumptive satellite cells and reinduces Erm expression in DRG-derived glia but not in Schwann cells from sciatic nerve. These data demonstrate that there are intrinsic differences between these glial subtypes in their response to NRG1 signaling. In neural crest cultures, Erm-positive progenitor cells give rise to two distinct glial subtypes: Erm-positive, Oct-6-negative satellite glia in response to GGF2, and Erm-negative, Oct-6-positive Schwann cells in the presence of serum and the adenylate cyclase activator forskolin. Thus, Erm-positive neural crest-derived progenitor cells and presumptive satellite glia are able to acquire Schwann cell features. Given the in vivo expression of Erm in peripheral ganglia, we suggest that ganglionic Erm-positive cells may be precursors of Schwann cells.