Increased connexin43 expression in human saphenous veins in culture is associated with intimal hyperplasia.

OBJECTIVE: Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. Connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. METHODS: Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis. RESULTS: Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. After 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. A time-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression. CONCLUSIONS: These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia. CLINICAL RELEVANCE: Stenosis due to intimal hyperplasia is the most common cause of failure of venous bypass grafts. To better understand the development of intimal hyperplasia, we used an ex vivo organ culture model to study saphenous veins harvested from patients undergoing a lower limb bypass surgery. In this model, the morphologic and functional integrity of the vessel wall is maintained and significant intimal hyperplasia development occurs after 14 days in culture. We have postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, may participate in the development of intimal hyperplasia. Indeed, intimal hyperplasia consists of proliferation and migration of smooth muscle cells into the subendothelial space. Intercellular communication is responsible for the direct transfer of ions and small molecules from one cell to the other through gap-junction channels found at cell-cell appositions. No study to date has evaluated whether gap junctional communication is involved in the process of intimal hyperplasia in humans. This assertion was investigated by using the aforementioned organ culture model of intimal hyperplasia in human saphenous veins, and our data support a critical role for Cx43-mediated gap junctional communication in human vein during the development of intimal hyperplasia.

Dexamethasone stimulates the biochemical differentiation of fetal forebrain cells in reaggregating cultures.

The influence of dexamethasone on the development of neurons and oligodendrocytes was studied in serum-free, aggregating rat brain cell cultures. Synaptogenesis and myelination occur in this culture system. The concentration of myelin basic protein and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase were used as oligodendroglia and myelin markers. Choline acetyltransferase and acetylcholinesterase served as neuronal markers, glutamine synthetase reflected astrocyte differentiation, while ornithine decarboxylase served as a general marker for cell growth and maturation. This study showed that dexamethasone stimulated the differentiation of cholinergic neurons and astrocytes. The effect of dexamethasone on oligodendroglial differentiation and myelination depended on the stage of development: during the early phase of myelination dexamethasone had a stimulatory effect, whereas at a later stage it showed a significant inhibition.

Personality and Culture Cross-Cultural Psychology at the Next Crossroads

A review of nearly three decades of cross-cultural research shows that this domain still has to address several issues regarding the biases of data collection and sampling methods, the lack of clear and consensual definitions of constructs and variables, and measurement invariance issues that seriously limit the comparability of results across cultures. Indeed, a large majority of the existing studies are still based on the anthropological model, which compares two cultures and mainly uses convenience samples of university students. This paper stresses the need to incorporate a larger variety of regions and cultures in the research designs, the necessity to theorize and identify a larger set of variables in order to describe a human environment, and the importance of overcoming methodological weaknesses to improve the comparability of measurement results. Cross-cultural psychology is at the next crossroads in it's development, and researchers can certainly make major contributions to this domain if they can address these weaknesses and challenges.

Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity : a role for microglia

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

Bacterial colonization and infection of electrophysiological cardiac devices detected with sonication and swab culture.

BACKGROUND: Electrophysiological cardiac devices are increasingly used. The frequency of subclinical infection is unknown. We investigated all explanted devices using sonication, a method for detection of microbial biofilms on foreign bodies. METHODS AND RESULTS: Consecutive patients in whom cardiac pacemakers and implantable cardioverter/defibrillators were removed at our institution between October 2007 and December 2008 were prospectively included. Devices (generator and/or leads) were aseptically removed and sonicated, and the resulting sonication fluid was cultured. In parallel, conventional swabs of the generator pouch were performed. A total of 121 removed devices (68 pacemakers, 53 implantable cardioverter/defibrillators) were included. The reasons for removal were insufficient battery charge (n=102), device upgrading (n=9), device dysfunction (n=4), or infection (n=6). In 115 episodes (95%) without clinical evidence of infection, 44 (38%) grew bacteria in sonication fluid, including Propionibacterium acnes (n=27), coagulase-negative staphylococci (n=11), Gram-positive anaerobe cocci (n=3), Gram-positive anaerobe rods (n=1), Gram-negative rods (n=1), and mixed bacteria (n=1). In 21 of 44 sonication-positive episodes, bacterial counts were significant (>or=10 colony-forming units/mL of sonication fluid). In 26 sterilized controls, sonication cultures remained negative in 25 cases (96%). In 112 cases without clinical infection, conventional swab cultures were performed: 30 cultures (27%) were positive, and 18 (60%) were concordant with sonication fluid cultures. Six devices and leads were removed because of infection, growing Staphylococcus aureus, Streptococcus mitis, and coagulase-negative staphylococci in 6 sonication fluid cultures and 4 conventional swab cultures. CONCLUSIONS: Bacteria can colonize cardiac electrophysiological devices without clinical signs of infection.

Raja-Yoga : Histoire et tradition. Vol 7 : Glossaire de culture indienne

Voici rassemblées en un ouvrage les philosophies, les origines et les filiations du Yoga, plus particulièrement du Raja-Yoga, dans une vision plurielle et chronologique. Le lecteur au fil des textes sélectionnés prend conscience des moyens extraordinaires et exigeants développés par les sages de l'Inde pour répondre aux questions universelles du sens de la vie et de sa finalité. À l'opposé de l'esprit occidental, qui s'appuie sur la dialectique pour explorer le monde, le Yoga se défie du seul intellect, et privilégie l'expérience de soi. Les témoignages et les révélations d'êtres spirituels, tels que Patanjali, Vivekananda ou Ram Chandra, montrent que le chemin de la connaissance passe par soi, le chercheur étant à la fois l'objet de l'expérience, l'observateur et la conscience transformée. S'adressant à chacun de nous dans sa quête de sens et de vérité, le Raja-Yoga est une science, une méthode de réalisation de soi vivante qui, tout en s'inscrivant dans une tradition religieuse et philosophique, reste libre d'évoluer et de répondre aux besoins de l'homme à chaque époque. Ce coffret a eu pour désir de mettre à disposition d'un public occidental la spiritualité de l'Inde vue par elle-même, sous l'angle du Raja-Yoga. L'intérêt porté par des universitaires occidentaux à cette démarche a contribué à mettre en valeur l'apport remarquable des chercheurs indiens, praticiens et érudits, autour d'un sujet sur lequel il n'existait pas d'étude approfondie. Pour la première fois, le Raja-Yoga révèle sa noblesse et son efficacité dans sa dimension de science spirituelle ouverte à tous.

Glutamine Synthetase is Expressed by Primary Sensory Neurons from Chick Embryos In Vitro but not In Vivo: Influence of Skeletal Muscle Extract.

Glutamine synthetase (GS) catalyses the ATP-dependent formation of glutamine from glutamate and ammonia. To determine whether dorsal root ganglion (DRG) cells from chick embryos express the enzyme in vivo or in vitro, GS was detected by immunocytochemical reaction either in vibratome sections of DRG or in dissociated DRG cell cultures. The immunocytochemical detection of GS showed that in vivo the DRG taken from chick embryos at day 10 (E10), E14, E18 or from chickens after hatching were free of any GS-positive ganglion cells; in contrast, in neuron-enriched cultures of DRG cells grown in vitro at E10, virtually all the neuronal cells (98.6 +/- 1.0%) express GS at 3, 5 or 7 days of culture. In mixed DRG cell cultures, only 83.6+/-4.6% of the neurons displayed a GS-immunoreactivity. In both culture conditions, neither the presence of horse serum nor the age of the culture appeared to affect the percentage of neurons which displayed a GS-immunoreactivity. After [3H]glutamine uptake, radioautographs revealed that only 80% of the neurons were labelled in neuron-enriched DRG cell cultures while 96% of the neurons were radioactive in mixed DRG cell cultures. Furthermore the most heavily [3H]glutamine-labelled neurons were exclusively found in mixed DRG cell cultures. Combination of both immunocytochemical detection of GS and radioautography after [3H]glutamine uptake showed that strongly GS-immunostained neurons corresponded to poorly radioactive ones and vice versa. When skeletal muscle extract (ME) was added to DRG cell cultures, the number of GS-positive neurons was reduced to 77.5 +/- 2.5% in neuron-enriched cultures or to 43.6 +/- 3.8% in mixed DRG cell cultures; in both types of culture, the intensity of the neuronal immunostaining was depressed. Furthermore, combined action of ME and non-neuronal cells potentiates the enzyme repression exerted separately by ME or non-neuronal cells. Since GS-immunoreactivity is expressed in DRG cells grown in vitro, but not in vivo, it is suggested that microenvironmental factors influence the expression of GS. More specifically, the repression of GS by primary sensory neurons grown in vitro may be strongly induced by soluble factors present in skeletal muscle, and to a lesser extent in brain, and potentiated by non-neuronal cells.

Involvement of autophagy in hypoxic-excitotoxic neuronal death.

Neuronal autophagy is increased in numerous excitotoxic conditions including neonatal cerebral hypoxia-ischemia (HI). However, the role of this HI-induced autophagy remains unclear. To clarify this role we established an in vitro model of excitotoxicity combining kainate treatment (Ka, 30 µM) with hypoxia (Hx, 6% oxygen) in primary neuron cultures. KaHx rapidly induced excitotoxic death that was completely prevented by MK801 or EGTA. KaHx also stimulated neuronal autophagic flux as shown by a rise in autophagosome number (increased levels of LC3-II and punctate LC3 labeling) accompanied by increases in lysosomal abundance and activity (increased SQSTM1/p62 degradation, and increased LC3-II levels in the presence of lysosomal inhibitors) and fusion (shown using an RFP-GFP-LC3 reporter). To determine the role of the enhanced autophagy we applied either pharmacological autophagy inhibitors (3-methyladenine or pepstatinA/E64) or lentiviral vectors delivering shRNAs targeting Becn1 or Atg7. Both strategies reduced KaHx-induced neuronal death. A prodeath role of autophagy was also confirmed by the enhanced toxicity of KaHx in cultures overexpressing BECN1 or ATG7. Finally, in vivo inhibition of autophagy by intrastriatal injection of a lentiviral vector expressing a Becn1-targeting shRNA increased the volume of intact striatum in a rat model of severe neonatal cerebral HI. These results clearly show a death-mediating role of autophagy in hypoxic-excitotoxic conditions and suggest that inhibition of autophagy should be considered as a neuroprotective strategy in HI brain injuries.

Phosphorylation of neurofilament subunit NF-M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape.

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists was due to phosphorylation. Neurons treated with cyclosporin, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, also showed increased levels of phosphorylated NF-M. Moreover, analysis of neurofilament stability revealed that the phosphorylation of NF-M, resulting from NMDA receptor inhibition, enhanced the solubility of NF-M. Finally, cortical neurons cultured in the presence of the NMDA receptor antagonists MK801 and AP5 grew longer neurites. Together, these data indicate that a blockade of NMDA receptors during development of cortical neurons increases the phosphorylation state and the solubility of NF-M, thereby favoring neurite outgrowth. This also underlines that dynamics of the neurofilament and microtubule cytoskeleton is fundamental for growth processes.

Oxygen tension controls the expression of the monocarboxylate transporter MCT4 in cultured mouse cortical astrocytes via a hypoxia-inducible factor-1α-mediated transcriptional regulation.

The monocarboxylate transporter MCT4 is a high capacity carrier important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is predominantly expressed by astrocytes. Surprisingly, MCT4 expression in cultured astrocytes is low, suggesting that a physiological characteristic, not met in culture conditions, is necessary. Here we demonstrate that reducing oxygen concentration from 21% to either 1 or 0% restored in a concentration-dependent manner the expression of MCT4 at the mRNA and protein levels in cultured astrocytes. This effect was specific for MCT4 since the expression of MCT1, the other astrocytic monocarboxylate transporter present in vitro, was not altered in such conditions. MCT4 expression was shown to be controlled by the transcription factor hypoxia-inducible factor-1α (HIF-1α) since under low oxygen levels, transfecting astrocyte cultures with a siRNA targeting HIF-1α largely prevented MCT4 induction. Moreover, the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced MCT4 expression in astrocytes cultured in presence of 21% oxygen. In parallel, glycolytic activity was enhanced by exposure to 1% oxygen as demonstrated by the increased lactate release, an effect dependent on MCT4 expression. Finally, MCT4 expression was found to be necessary for astrocyte survival when exposed for a prolonged period to 1% oxygen. These data suggest that a major determinant of astrocyte MCT4 expression in vivo is likely the oxygen tension. This could be relevant in areas of high neuronal activity and oxygen consumption, favouring astrocytic lactate supply to neurons. Moreover, it could also play an important role for neuronal recovery after an ischemic episode.

PROBLEMATIQUE DE LA CULTURE EN MEDECINE DE FAMILLE, UNE APPROCHE CENTREE SUR LE PATIENT

Problématique : En réaction à l'émergence de société plurielle, la communauté médicale a mis en place des outils de prise en charge de l'altérité. Cependant, la culture, issue notamment des origines, des croyances et des expériences est unique à chaque patient et lui sert à appréhender la maladie. Ce pourquoi, les outils de prise en compte de la culture, et de valorisation du patient, peuvent être potentiellement étendus à tous. Objectifs : Déterminer la place de la culture en médecine de famille, les enjeux qui en dépendent et la pertinence de l'utilisation des compétences transculturelles avec tous les patients. Perfectionner ces compétences dans le but de créer un modèle bio-psycho-socio-culturel améliorant la communication et la compréhension entre patients et thérapeutes. Méthodologie : Réalisation d'une revue de la littérature en déterminant comment la culture est intégrée à la médecine sous forme de savoir théorique. Réalisation d'une recherche qualitative de type exploratoire sur la base d'entretiens enregistrés auprès de 7 médecins de famille romands, pour montrer comment la notion de culture est intégrée en pratique. Comparaison entre médecine transculturelle et approche pratique de terrain. Résultats : La culture influe sur toutes les étapes de la consultation. La pratique des médecins est influencée par leur conception de la culture. La construction commune d'une représentation de la maladie, basée sur les conceptions du patient, améliore la collaboration thérapeutique. Les compétences cliniques transculturelles actuelles semblent être applicables à tous, mais nécessitent un léger développement en intégrant l'expérience pratique quotidienne de la médecine de famille. Conclusion : L'utilisation des compétences cliniques transculturelles (savoir, savoir-faire, savoir-être) avec tous les patients est réalisable et peut être affinée d'une quatrième compétence le savoir- percevoir : capacité d'être alerte et réceptif à toute subtilité culturelle qui ressurgit de l'interaction.

Comparative assessment of intimal hyperplasia development after 14 days in two different experimental settings: tissue culture versus ex vivo continuous perfusion of human saphenous vein.

BACKGROUND: Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise controlled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. MATERIALS AND METHODS: Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, alpha-actin, and PAI-l were determined before and after 14days of either experimental conditions. RESULTS: Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell (SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII, and alpha-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. CONCLUSION: This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

Substance P and calbindin D-28k-immunoreactivity in primary sensory neurons of chick embryos: differential neuronal birthdates and transient co-localization.

During the ontogenesis of dorsal root ganglia (DRG), the immunoreactivity to substance P (SP) and calbindin D-28k (CaBP) appears in chickens at embryonic day 5 (E5) and E10 respectively. To establish the birthdates of primary sensory neurons expressing SP or CaBP, chick embryos were given repetitive intra-amniotic injections of [3H]-thymidine. The neuroblasts giving rise to SP-expressing neurons were labeled up to E6 while those generating CaBP-immunoreactive neurons stopped to incorporate [3H]-thymidine before E5.5. This finding indicates that neurons exhibiting distinct phenotypes may originate from neuroblasts which arrest to proliferate at close but distinct stages of development. To determine whether SP and CaBP are co-expressed or not in DRG neurons, chick embryos at E12, E18, and chickens two weeks after hatching were perfused and fixed to detect simultaneously SP- and CaBP-immunoreactivity in DRG sections. The results showed that SP and CaBP were transiently co-expressed by a subset of neurons at E12. Later, however, the SP-immunoreactivity was gradually lost by these ganglion cells, so that the SP- and CaBP-immunoreaction defined two distinct neuronal subpopulations after hatching. In conclusion, most CaBP-immunoreactive DRG cells derive from a subset of neurons in which SP and CaBP are transiently co-localized.