Adherence to direct oral anticoagulant treatment for atrial fibrillation in the Netherlands: A surveillance study.

BACKGROUND: Adherence to direct oral anticoagulants (DOACs) in patients with atrial fibrillation in every day practice may be less than in clinical trials. AIMS: To assess adherence to DOACs in atrial fibrillation patients in every day practice and identify predictors for non-adherence. METHODS: Individual linked dispensing data of atrial fibrillation patients who used DOACs were obtained from the Foundation for Pharmaceutical Statistics covering the Netherlands between 2012 and 2016. One year adherence to DOAC was calculated for initial DOAC as proportion of days covered (PDC) ≥80% and the association between clinical variables and adherence was assessed using logistic regression. In addition, we measured non-persistence, that is, patients who completely stopped their initial DOAC within 1 year follow-up. RESULTS: A total of 4797 apixaban-, 20 454 rivaroxaban- and 18 477 dabigatran users were included. The mean age was 69 years (n = 43 910), which was similar for the DOAC types. The overall proportion of patients with PDC ≥80% was 76%, which was highest for apixaban- (87%), followed by dabigatran- (80%) and rivaroxaban (69%) users. Multivariable analyses revealed that age ≤60 years, no concomitant drug use were predictors for non-adherence. Of atrial fibrillation patients who continued treatment, 97% had a PDC ≥80%, compared with only 56% for those who discontinued their DOAC treatment within 1 year. CONCLUSIONS: Non-adherence to DOACs was associated with age ≤60 years and no concomitant drugs use. Non-adherence was higher in patients who later discontinued DOAC treatment. Results of our study support research into interventions to improve adherence.

Vitamin K1 supplementation to improve the stability of anticoagulation therapy with vitamin K antagonists: a dose-finding study.

BACKGROUND: Poor anticoagulant stability in patients using vitamin K antagonists is a risk factor for both bleeding and thrombosis. In previous studies supplementation with low dose vitamin K(1) was shown to improve the stability of anticoagulant control. We set up a study to confirm earlier reports and to determine the optimal daily dose of vitamin K(1) in preparation of a large study with clinical endpoints. DESIGN AND METHODS: Four hundred patients from two anticoagulation clinics starting with vitamin K antagonists, independently of a possible history of instable anticoagulation, were randomized to receive either placebo or 100, 150 or 200 µg of vitamin K(1) together with their treatment with vitamin K antagonists. The treatment was administered for 6 to 12 months. Anticoagulation stability, expressed as the percentage of time that the International Normalized Ratio was within the therapeutic range, was compared between the groups. RESULTS: After adjustment for age, sex, vitamin K antagonist used, anticoagulation clinic and interacting drugs as confounding factors the difference in percentage of time with the International Normalized Ratio within the therapeutic range between the placebo group and the vitamin K(1) groups was 2.1% (95% CI: -3.2% - 7.4%) for the group taking 100 µg, 2.7% (95% CI: -2.3% -7.6%) for the group taking 150 µg and 0.9% (95% CI: -4.5% - 6.3%) for the group taking 200 µg vitamin K(1) group, in favor of the vitamin K(1) groups. The patients from both the 100 µg group and the 150 µg group had a 2-fold higher chance of reaching at least 85% of time with the International Normalized Ratio within the therapeutic range. There were no differences in thromboembolic or hemorrhagic complications between the groups. CONCLUSIONS: In patients starting vitamin K antagonists, supplementation with low dose vitamin K(1) resulted in an improvement of time that anticoagulation was within the therapeutic range. Differences between doses were, however, small and the improvement is unlikely to be of clinical relevance. For future studies we recommend selecting only patients with instable anticoagulant control. (This study was registered at www.isrctn.org as ISRCTN37109430).

L1 and laminin: their expression during rat hypophysis ontogenesis and in adult neurohemal areas.

L1 is a murine multidomain glycoprotein implicated in cell aggregation, fasciculation. neurite outgrowth and synaptogenesis. Laminin, a trimeric polypeptide, is implicated in neuronal survival, growth cone guidance, neurite outgrowth and cell differentiation. Laminin can also interact with the cell adhesion molecule L1. Their expressions were investigated from embryonic day 15 (E15) to adult in the rat hypophysis, and in adult neurohemal zones. Detected in the neural lobe from E17, the L1 immunoreactivity increased during prenatal development and persisted in adulthood mainly related to the neuropeptidergic fibers. Pituicytes were only labelled on the plasmalemma apposed to axons. In the intermediate lobe, L1 appeared at birth on folliculo-stellate cells extensions, constituting a network which densified during postnatal development. L1 is also expressed in all neurohemal areas on neuronal profiles. Laminin was clearly detectable in the hypophysis at E15 before the first blood vessels penetrate the Rathke pouch. At E20, all the basal membranes of the blood vessels were stained. In the intermediate lobe, a spotted laminin immunoreactivity was detected at E21. At this stage, we observed the staining of intercellular spaces and the intracellular labelling of melanotrophs, concerning reticulum or vesicles. The staining of melanotrophs seemed to maintain during adulthood. In contrast with blood vessels of the adult cerebral tissue, adult capillaries of the neural lobe and the others neuro-hemal zones were intensely labelled with the anti-laminin antibody. These results suggest that neurite outgrowth and neurite guidance could be promoted by L1 and laminin in the neurointermediate lobe. The "intercellular tunnels" could also be an important guidance cue for migrating cells in the intermediate lobe. These data also demonstrate that melanotrophic cells. secreting the laminin, have a role in the ontogenesis of the gland. Finally, we suggest that L1 and laminin can collaborate to reinforce "neurons-capillaries" interactions in neurohemal zones.

Differentiation of rat hypothalamic dopaminergic neurons is stimulated in vitro by target cells: the melanotrophs.

We have investigated in vitro the influence of pituitary intermediate lobe melanotrophs on the differentiation of their afferent hypothalamic dopaminergic neurons. The presence of melanotrophs in primary cultures of foetal hypothalamic neurons induces an increase of the number of dopaminergic neurons (while the total neuronal population remains unchanged) and induces a stimulation of their neuritic outgrowth. These effects are mediated by diffusible factors since they are reproduced by application of conditioned medium issued from co-cultures with intermediate lobe cells from newborn rats. Moreover, by immunoneutralization of alpha-melanocyte-stimulating hormone (alphaMSH) in the co-culture or conditioned medium, or by application of the peptide itself, we demonstrate that the neuritotrophic effect on dopaminergic neurons is mediated by alphaMSH, the main secretory product of melanotrophs, whereas the inductive effect on the number of dopaminergic neurons is attributable to another diffusible neurotrophic factor(s) present in foetal, but not adult, adenohypophysis. Similar effects are observed on cultures of newborn hypothalamic neurons. However, at this stage of neuronal development, alphaMSH also increases the number of dopaminergic neurons, which could be due to a change of neuronal receptivity. We show that the neuritotrophic influence of alphaMSH is restricted to the dopaminergic neurons connected to the melanotrophs, and that in addition, these neurons systematically co-express the tyrosine hydroxylase and glutamate decarboxylase as the neurons innervating the melanotrophs in situ. These findings indicate that the differentiation of dopaminergic hypothalamic neurons is influenced by the target cells, melanotrophs, and that this trophic influence implicates alphaMSH.

Co-culture of hypothalamic neurons and melanotrope cells: a model to study synaptogenesis between central neurons and endocrine cells.

As a first step towards elucidating mechanisms involved in neuroendocrine synaptogenesis, we developed a model of co-culture based on hypothalamic-intermediate pituitary interactions. Dissociated hypothalamic neurons from fetal rats at embryonic day 15 were cultured in a defined medium together with melanotrope cells of the pituitary intermediate lobe from neonatal rats. In these co-cultures, establishment of synaptic contacts between GABAergic or dopaminergic neurons and an endocrine target cell the melanotrope cell, was studied by morphofunctional approaches. Using double immunostaining with antibodies directed against glutamate decarboxylase or tyrosine hydroxylase and alpha-melanocyte-stimulating hormone, we demonstrated morphological contacts between GABAergic or dopaminergic neurons and melanotrope cells as early as three days in vitro. Furthermore, using an antibody directed against synapsin I, we showed a modification of synapsin I immunoreactivity from diffuse to punctate distribution correlated with the establishment of contacts and the observation of characteristic neuroendocrine synapses by electron microscopy. These results were further confirmed by electrophysiological studies. Patch-clamp recordings demonstrated that, at six days in vitro, some melanotrope cells displayed GABAergic synaptic currents, which occurred either spontaneously and/or could be evoked chemically by 50 mM KCl or 100 microM kainate. The proportion of the melanotrope cells receiving functional synaptic inputs increased until 10 days in culture, a stage at which virtually all melanotrope cells in contact with neurons possessed functional synapses. The results presented here describe the establishment of neuroendocrine synapses in vitro, studied by combining morphofunctional and electrophysiological approaches.

Expression of the c-ets1 gene in the hypothalamus and pituitary during rat development.

The Ets gene family codes for transcription factors containing a conserved DNA binding domain: the Ets-binding domain. The proto-oncogene c-ets1 is highly expressed in lymphoid organs and in developing mesodermal-originating structures. We studied c-ets1 gene expression in the developing rat hypothalamo-hypophyseal system, using in situ hybridization on paraformaldehyde-fixed frozen sections. At embryonic day 12 (E12) and E13, cells synthesizing c-ets1mRNA are found in the neural tube where they form small, heavily labeled strand-like and punctate structures; positive mesenchymatous cells, corresponding to the surface capillary network, surround the brain and hypophysis. C-ets1mRNA is synthesized from E14 in the neural pituitary and E15 in the adenohypophysis, during angiogenesis; no c-ets1mRNA is detected in the avascular intermediate pituitary at any stage. Strand-like c-ets1mRNA labeling is intense from E14 to E21 in the diencephalon. This labeling is also detected during perinatal stages in the hypothalamic magnocellular nuclei, one of the most richly vascularized brain areas. In the rat hypothalamo-hypophyseal system, c-ets1 gene expression is maximal during fetal and perinatal stages and progressively decreases thereafter until adulthood. The spatio-temporal correlation observed between c-ets1 gene expression and blood vessel formation in the rat hypothalamus and pituitary suggests a role for c-ets1 in angiogenesis in this system.

Characterization of functional GABAergic synapses formed between rat hypothalamic neurons and pituitary intermediate lobe cells in coculture: Ca2+ dependence of spontaneous IPSCs.

Rat hypothalamic neurons and endocrine cells from the intermediate lobe of the pituitary were grown in dissociated coculture. Neurons positively stained with an antibody against glutamate decarboxylase established apparent contacts with the alpha-melanocyte-stimulating hormone-positive endocrine cells. These sites of contact were intensely labeled with an antibody against the synaptic protein synapsin I and displayed ultrastructural features characteristic of synapses. Using patch-clamp recordings, we have demonstrated that these contacts correspond to functional GABAergic synapses. The synaptic currents were blocked reversibly by bicuculline (5 microM) and SR95531 (5 microM), two competitive antagonists of the GABAA receptor. At a holding potential of -60 mV, spontaneously occurring IPSCs (s-IPSCs) had small amplitudes (10-100 pA), whereas electrically evoked IPSCs (ee-IPSCs) had amplitudes up to 1 nA. The rise times of both types of IPSCs were fast ( < or = 1 msec), and their decaying phases were fitted in most cases with a single exponential function (time constant 50 msec). The amplitude distribution of s-IPSCs did not reveal clear, equally spaced peaks and was little affected by tetrodotoxin, suggesting that most s-IPSCs were miniature IPSCs. Reduction of extracellular calcium concentration to 0.3 mM induced a marked decrease in s-IPSC frequency and revealed a single amplitude peak at 10 pA, suggesting that a single quantum of GABA activates 8-10 GABAA channels. Thus, our preparation might be an interesting model to study different aspects of synapse formation between a central neuron and its target as well as the fundamental mechanisms of synaptic transmission at central synapses.

Ontogeny of prodynorphin gene expression in the rat hypothalamus.

Opioid peptides, deriving from prodynorphin, proenkephalin and proopiomelanocortin genes, have been shown to modulate brain development. Prodynorphin gene expression was studied here by in situ hybridization in the developing rat hypothalamus using oligodeoxynucleotide probes. Prodynorphin mRNA-synthetizing cells were observed in the ventromedial hypothalamic nucleus, the supraoptic and the paraventricular nuclei from embryonic days 16, 18 and 21, respectively. We detected no transient expression of prodynorphin gene in the rat hypothalamus. Prodynorphin mRNA-containing cells were also observed prenatally in the striatum, the cortex, the hippocampus and the amygdala. When compared with data from the literature, our results suggest that translation may immediately follow transcription of prodynorphin gene in the supraoptic nucleus. The presence of prodynorphin mRNA in the developing rat hypothalamus also raises the possibility of an involvement of prodynorphin-derived peptides in developmental processes and/or in the maturation of adult neural regulations.

Ontogeny of proenkephalin gene expression in the rat hypothalamus.

In the rat hypothalamus, proenkephalin (PE) mRNA synthetizing cells were detected by in situ hybridization, using synthetic oligodeoxy-nucleotides, from embryonic day 14 (E14) in the presumptive anterior hypothalamic area (AHA) and preoptic part of the bed nucleus of the stria terminalis (BST), and from E18 in the developing median preoptic area, perifornical area, suprachiasmatic nucleus, dorsomedial and ventromedial hypothalamic nuclei. In the paraventricular nucleus, cells expressed PE gene in the late prenatal stages; both parvo- and magnocellular neurons synthetized PEmRNA in the early postnatal stages. Cells expressing PE gene were observed after birth in the lateral preoptic area, lateral hypothalamus, medial and lateral parts of the BST. PEmRNA was also found from E14 in the striatum, from E18 in the central and medial amygdaloid nuclei, the medial group of the thalamic nuclei, and postnatally in a second more anterior structure of the thalamus. In the hypothalamus, a clear similarity was observed between adult and developmental distributions of PE gene expressing cells. The early onset of PE gene expression in the developing rat diencephalon suggests an involvement of PE in developmental processes, such as cell proliferation and differentiation; the presence of PE during the perinatal period may also indicate the appearance of adult neural regulations.

In situ localization of NF-H neurofilament subunit mRNAs in rat brain.

The expression of NF-H neurofilament subunit mRNAs was investigated in the rat brain at different ontogenic stages. The levels of NF-H mRNAs vary 15-fold among brain regions with the highest level in the brainstem. In situ localization studies revealed that the NF-H mRNAs are mainly concentrated in the brainstem motoneuron nuclei. By increasing the sensitivity of the hybridization method, NF-H mRNAs could also be localized in neurons present in the cortex, thalamus and hippocampus areas. Minor amounts of NF-H mRNAs were already detected at 17-day embryonic stages.

Expression of the oxytocin and vasopressin genes in the rat hypothalamus during development: an in situ hybridization study.

Oxytocin (OT)- and vasopressin (VP)-mRNAs were detected in the hypothalamus, during development, by in situ hybridization with synthetic oligonucleotide probes. The presence of VP- and OT-mRNAs was first detected in the supraoptic nucleus at E16 and E17 respectively, and simultaneously at E18 in the paraventricular nucleus. VP- (but not OT-) mRNA then appeared in the suprachiasmatic nucleus at E21, and OT- (but not VP-) mRNA, in the anterior commissural nucleus at time of birth. In the different nuclei, the relative distribution of cells containing OT- or VP-mRNA was comparable, from the earliest stages on, with that observed in the adult. These data suggest that the later appearance of mature OT (E20), versus VP (E16), reported in immunocytochemical studies, may not be due to a delayed transcription. Moreover, since the presence of both OT- and VP-prohormones has been reported at E16, the results support the idea of a rapid translation of both OT- and VP-mRNAs. In no location could OT- and VP-mRNAs be detected before final cell settlement; the possible role of environmental factors in final non-proliferative differentiation is discussed.