Hypergravity Increases Blood-Brain Barrier Permeability to Fluorescent Dextran and Antisense Oligonucleotide in Mice.

The earliest effect of spaceflight is an alteration in vestibular function due to microgravity. Hypergravity exposure induced by centrifugation is also able to provoke motion sickness. The blood-brain barrier (BBB) is the crucial interface between the vascular system and the brain to ensure efficient neuronal activity. We developed experimental protocols of hypergravity on C57Bl/6JRJ mice to induce motion sickness and reveal its effects on the BBB. Mice were centrifuged at 2× g for 24 h. Fluorescent dextrans with different sizes (40, 70 and 150 kDa) and fluorescent antisense oligonucleotides (AS) were injected into mice retro-orbitally. The presence of fluorescent molecules was revealed by epifluorescence and confocal microscopies in brain slices. Gene expression was evaluated by RT-qPCR from brain extracts. Only the 70 kDa dextran and AS were detected in the parenchyma of several brain regions, suggesting an alteration in the BBB. Moreover, Ctnnd1, Gja4 and Actn1 were upregulated, whereas Jup, Tjp2, Gja1, Actn2, Actn4, Cdh2 and Ocln genes were downregulated, specifically suggesting a dysregulation in the tight junctions of endothelial cells forming the BBB. Our results confirm the alteration in the BBB after a short period of hypergravity exposure.

Effects of centrifugation and whole-body vibrations on blood-brain barrier permeability in mice.

Modifications of gravity levels induce generalized adaptation of mammalian physiology, including vascular, brain, muscle, bone and immunity functions. As a crucial interface between the vascular system and the brain, the blood-brain barrier (BBB) acts as a filter to protect neurons from pathogens and inflammation. Here we compare the effects of several protocols of hypergravity induced by centrifugation and whole-body vibrations (WBV) on BBB integrity. The immunohistochemistry revealed immunoglobulin G (IgG) extravasation from blood to hippocampal parenchyma of mice centrifuged at 2 × g during 1 or 50 days, whereas short exposures to higher hypergravity mimicking the profiles of spaceflight landing and take-off (short exposures to 5 × g) had no effects. These results suggest prolonged centrifugation (>1 days) at 2 × g induced a BBB leakage. Moreover, WBV were similarly tested. The short exposure to +2 × g vibrations (900 s/day at 90 Hz) repeated for 63 days induced IgG extravasation in hippocampal parenchyma, whereas the progressive increase of vibrations from +0.5 to +2 × g for 63 days was not able to affect the IgG crossing through the BBB. Overall, these results suggest that the BBB permeability is sensitive to prolonged external accelerations. In conclusion, we advise that the protocols of WBV and centrifugation, proposed as countermeasure to spaceflight, should be designed with progressively increasing exposure to reduce potential side effects on the BBB.

[Use of placebo in French hospitals: data from polyvalent medicine units]. / Le placebo à l'hôpital - Regard sur son utilisation dans les services de médecine polyvalente.

A placebo drug is defined as a treatment without any specific pharmacological efficacy, that works when the patient thinks to receive an active treatment, through a psychological and physiological mechanism. This study aimed to evaluate the use of placebo in French hospitals, in Polyvalent Medicine units. A questionnaire comprising 15 items was sent to 372 units. The analysis of 153 responses was conducted from dynamic crosstabs in Excel and using the R software available online. The survey confirmed that the use of placebos in hospital is frequent, with nearly 2/3 of professionals answering the questionnaire declared to use it. The oral capsule is the most commonly used form. Placebo is mainly administered at night, in case of pain, insomnia or anxiety, to so-called "difficult" patients. Placebo is not always given after medical prescription. In most cases, patients are not informed that they receive a placebo. The majority of professionals believed in the placebo effect but considered to be insufficiently informed and trained in the use of placebo in current practice. Although the placebo effect is now demonstrated, ethical and legal considerations recommend placebo treatment only on medical prescription, with the prior information of the patient. The placebo could be used as complementary therapy to conventional treatment in the cases of this therapeutic effectiveness has been demonstrated. Professionals should be trained in the use of placebo in order to avoid nocebo effect and potentiate beneficial effects of placebo.

TITLE: Le placebo à l'hôpital - Regard sur son utilisation dans les services de médecine polyvalente. ABSTRACT: Un placebo est défini comme un traitement sans efficacité pharmacologique propre qui agit, lorsque le patient pense recevoir un traitement actif, par des mécanismes psychologiques et physiologiques. Notre étude avait pour but d'évaluer l'utilisation du placebo dans les unités de médecine polyvalente des hôpitaux français. Pour cela, un questionnaire comprenant 15 items a été transmis à plusieurs unités. L'analyse des réponses reçues confirme que l'utilisation de placebos à l'hôpital est fréquente, près des deux tiers des professionnels ayant répondu déclarent en faire usage. L'administration du placebo en gélule par voie orale est la forme la plus couramment utilisée. À l'hôpital, il est administré principalement la nuit, le plus souvent sans prescription médicale, en cas de douleur, d'insomnie ou d'anxiété, à des patients dits « difficiles ¼ (solliciteurs d'attention et de soin). Dans la plupart des cas, les patients ne sont pas informés qu'il s'agit d'un placebo. La majorité des professionnels « croit ¼ en l'effet placebo, mais se considèrent cependant insuffisamment informés et formés à son utilisation. Fréquemment utilisé à l'hôpital, son efficacité thérapeutique étant largement admise, des considérations éthiques et juridiques imposent de recommander que sa nature soit précisée au patient lors de sa prescription (au même titre que les autres préparations hospitalières). On parlera alors de « placebo ouvert ¼, c'est-à-dire que des explications sur les effets et les mécanismes d'action du placebo seront données au patient. Des études récentes montrent que dire au patient qu'il reçoit un placebo ouvert n'affecte en rien son effet.

Endocytosis of Activated Muscarinic m2 Receptor (m2R) in Live Mouse Hippocampal Neurons Occurs via a Clathrin-Dependent Pathway.

Our aim was to examine the dynamics of the muscarinic m2 receptor (m2R), a G-protein coupled receptor (GPCR), after agonist activation in living hippocampal neurons, and especially clathrin dependency endocytosis. We have previously shown that the m2R undergoes agonist-induced internalization in vivo. However, the nature of the endocytotic pathway used by m2R after activation is still unknown in living neurons. Using live cell imaging and quantitative analyses, we have monitored the effect of stimulation on the fate of the membrane-bound m2R and on its redistribution in intraneuronal compartments. Shortly (6 min) after activation, m2R is internalized into clathrin immunopositive structures. Furthermore, after clathrin-dependent endocytosis, m2R associates with early and late endosomes and with subcellular organelles involved in degradation. Together, these results provide, for the first time, a description of m2R trafficking in living neurons and prove that m2R undergoes clathrin-dependent endocytosis before being degraded.

Murine splenic B cells express corticotropin-releasing hormone receptor 2 that affect their viability during a stress response.

Chronic stress is now recognized as a risk factor for disease development and/or exacerbation. It has been shown to affect negatively the immune system and notably the humoral immune response. Corticotropin-releasing hormone (CRH) is known to play a crucial role in stress response. CRH receptors are expressed on different immune cells such as granulocytes, monocytes and T cells. However, up to now, no CRH receptor has been described on B cells which are key players of the humoral immune response. In order to highlight new pathways by which stress may impact immunity, we investigated the role of CRH in B cells. Here we show that splenic B cells express the CRH receptor 2 (CRHR2), but not CRHR1. This receptor is functional since CRH treatment of B cells activates different signaling pathways (e.g. p38) and decreases B cell viability. Finally, we show that immunization of mice with two types of antigens induces a more intense CRHR staining in secondary lymphoid organs where B cells are known to respond to the antigen. Altogether our results demonstrate, for the first time, that CRH is able to modulate directly B cell activity through the presence of CRHR2.

Maillard reaction products from highly heated food prevent mast cell number increase and inflammation in a mouse model of colitis.

Links between food and inflammatory bowel diseases (IBDs) are often suggested, but the role of food processing has not been extensively studied. Heat treatment is known to cause the loss of nutrients and the appearance of neoformed compounds such as Maillard reaction products. Their involvement in gut inflammation is equivocal, as some may have proinflammatory effects, whereas other seem to be protective. As IBDs are associated with the recruitment of immune cells, including mast cells, we raised the hypothesis that dietary Maillard reaction products generated through heat treatment of food may limit the colitic response and its associated recruitment of mast cells. An experimental model of colitis was used in mice submitted to mildly and highly heated rodent food. Adult male mice were divided in 3 groups and received nonheated, mildly heated, or highly heated chow during 21 days. In the last week of the study, each group was split into 2 subgroups, submitted or not (controls) to dextran sulfate sodium (DSS) colitis. Weight variations, macroscopic lesions, colonic myeloperoxidase activity, and mucosal mast cell number were evaluated at the end of the experiment. Only highly heated chow significantly prevented DSS-induced weight loss, myeloperoxidase activity, and mast cell number increase in the colonic mucosa of DSS-colitic mice. We suggest that Maillard reaction products from highly heated food may limit the occurrence of inflammatory phases in IBD patients.

Blood brain barrier precludes the cerebral arteries to intravenously-injected antisense oligonucleotide.

Alternative splicing of the ryanodine receptor subtype 3 (RyR3) produces a short isoform (RyR3S) able to negatively regulate the ryanodine receptor subtype 2 (RyR2), as shown in cultured smooth muscle cells from mice. The RyR2 subtype has a crucial role in the control of vascular reactivity via the fine tuning of Ca(2+) signaling to regulate cerebral vascular tone. In this study, we have shown that the inhibition of RyR3S expression by a specific antisense oligonucleotide (asRyR3S) was able to increase the Ca(2+) signals implicating RyR2 in cerebral arteries ex vivo. Moreover, we tried to inhibit the expression of RyR3S in vivo. The asRyR3S was complexed with JetPEI and injected intravenously coupled with several methods known to induce a blood brain barrier disruption. We tested solutions to induce osmotic choc (mannitol), inflammation (bacteria lipopolysaccharide and pertussis toxin), vasoconstriction or dilatation (sumatriptan, phenylephrine, histamine), CD73 activation (NECA) and lipid instability (Tween80). All tested technics failed to target asRyR3 in the cerebral arteries wall, whereas the molecule was included in hepatocytes or cardiomyocytes. Our results showed that the RyR3 alternative splicing could have a function in cerebral arteries ex vivo; however, the disruption of the blood brain barrier could not induce the internalization of antisense oligonucleotides in the cerebral arteries, in order to prove the function of RYR3 short isoform in vivo.

A focus on mast cells and pain.

Mast cells (MCs) are immunocytes with secretory functions that act locally in peripheral tissues to modulate local hemodynamics, nociceptor activation and pain. They are also able to infiltrate the central nervous system (CNS), especially the spinal cord and the thalamus, but their cerebral function remains an enigma. A role in regulating the opening of the blood-brain barrier has been proposed. Paracrine-like action of MCs on synaptic transmission might also signal a modulation of the nervous system by the immune system. In this review, we examine the link between MCs and nociceptive process, at the periphery as well as in the CNS.

Decrease of cerebral mast cell degranulation after systemic administration of lipopolysaccharide.

INTRODUCTION: The mast cell is an immunocyte, but its functions in the brain remain unclear. MATERIALS AND METHODS: In a rat model of weak inflammation, we analyzed the effect of a gram-negative bacterial lipopolysaccharide injection (100 µg/kg) on thalamic mast cell (MC) population. CONCLUSION: We demonstrate a significant decrease of their degranulation, which suggests the implication of MC in preventing sepsis on the brain.

Histamine H(3) receptor-mediated signaling protects mice from cerebral malaria.

BACKGROUND: Histamine is a biogenic amine that has been shown to contribute to several pathological conditions, such as allergic conditions, experimental encephalomyelitis, and malaria. In humans, as well as in murine models of malaria, increased plasma levels of histamine are associated with severity of infection. We reported recently that histamine plays a critical role in the pathogenesis of experimental cerebral malaria (CM) in mice infected with Plasmodium berghei ANKA. Histamine exerts its biological effects through four different receptors designated H1R, H2R, H3R, and H4R. PRINCIPAL FINDINGS: In the present work, we explored the role of histamine signaling via the histamine H3 receptor (H3R) in the pathogenesis of murine CM. We observed that the lack of H3R expression (H3R(-/-) mice) accelerates the onset of CM and this was correlated with enhanced brain pathology and earlier and more pronounced loss of blood brain barrier integrity than in wild type mice. Additionally tele-methylhistamine, the major histamine metabolite in the brain, that was initially present at a higher level in the brain of H3R(-/-) mice was depleted more quickly post-infection in H3R(-/-) mice as compared to wild-type counterparts. CONCLUSIONS: Our data suggest that histamine regulation through the H3R in the brain suppresses the development of CM. Thus modulating histamine signaling in the central nervous system, in combination with standard therapies, may represent a novel strategy to reduce the risk of progression to cerebral malaria.

[Role of histamine and histamine receptors in the pathogenesis of malaria]. / Rôle de l'histamine et des récepteurs histaminiques dans la pathogenèse du paludisme.

A hallmark of the host response to Plasmodium parasite is an inflammatory reaction characterized by elevated histaminemia levels. Since histamine, which acts through four different receptors and which synthesis is under the control of the histidine decarboxylase (HDC), is endowed with pro-inflammatory and immunosuppressive activities, we hypothesized that this vaso-active amine may participe to malaria pathogenesis. Combining genetic and pharmacologic approaches by using H1R(-/-), H2R(-/-), H3R(-/-), HDC(-/-) mice and H1R, H2R-, and H3R-antagonists, respectively, we found that cerebral malaria-associated pathogenetic processes such as blood brain barrier disruption, and T lymphocyte sequestration to cerebral vascular endothelium in mice were associated with histamine production. The identification of this novel inflammatory pathway and its implication in Plasmodium infection may lead to novel strategies to manipulate the anti-Plasmodium immune response and may provide new therapeutic tools to alleviate malaria disease.

Mesenchymal stem cells induce a weak immune response in the rat striatum after allo or xenotransplantation.

Mesenchymal stem cells (MSCs) have attracted attention for their potential use in regenerative medicine such as brain transplantation. As MSCs are considered to be hypoimmunogenic, transplanted MSCs should not trigger a strong host inflammatory response. To verify this hypothesis, we studied the brain immune response after transplantation of human or rat MSCs into the rat striatum and MSC fate at days 5, 14, 21 and 63 after transplantation. Flow cytometry analysis indicated that both MSCs express CD90 and human leucocyte antigen (MHC) class I, but no MHC class II molecules. They do not express CD45 or CD34 antigens. However, MSC phenotype varies with passage number. Human MSCs have mRNAs for interleukin (IL)-6, IL-8, IL-12, tumour necrosis factor (TNF)-alpha and TGF-beta(1), whereas rat MSCs express IL-6-, IL-10-, IL-12- and TGF-beta(1)-mRNAs. The quantification shows higher levels of mRNAs for the anti-inflammatory molecules IL-6 and TGF-beta(1) than for pro-inflammatory cytokines IL-8 and IL-12; ELISA analysis showed no IL-12 whereas TGF-beta(1) and IL-6 were detected. Transplant size did not significantly vary between 14 and 63 days after transplantation, indicating an absence of immune rejection of the grafts. Very few mast cells and moderate macrophage and microglial infiltrations, observed at day 5 remained stable until day 63 after transplantation in both rat and human MSC grafts. The observations of very few dendritic cells, T alphabeta-cells, and no T gammadelta-lymphocytes, all three being associated with Tp rejection in the brain, support the contention that MSCs are hypoimmunogenic. Our results suggest that MSCs are of great interest in regenerative medicine in a (xeno)transplantation setting.

Inhibition of histamine-mediated signaling confers significant protection against severe malaria in mouse models of disease.

From the inoculation of Plasmodium sporozoites via Anopheles mosquito bites to the development of blood-stage parasites, a hallmark of the host response is an inflammatory reaction characterized by elevated histamine levels in the serum and tissues. Given the proinflammatory and immunosuppressive activities associated with histamine, we postulated that this vasoactive amine participates in malaria pathogenesis. Combined genetic and pharmacologic approaches demonstrated that histamine binding to H1R and H2R but not H3R and H4R increases the susceptibility of mice to infection with Plasmodium. To further understand the role of histamine in malaria pathogenesis, we used histidine decarboxylase-deficient (HDC(-/-)) mice, which are free of histamine. HDC(-/-) mice were highly resistant to severe malaria whether infected by mosquito bites or via injection of infected erythrocytes. HDC(-/-) mice displayed resistance to two lethal strains: Plasmodium berghei (Pb) ANKA, which triggers cerebral malaria (CM), and Pb NK65, which causes death without neurological symptoms. The resistance of HDC(-/-) mice to CM was associated with preserved blood-brain barrier integrity, the absence of infected erythrocyte aggregation in the brain vessels, and a lack of sequestration of CD4 and CD8 T cells. We demonstrate that histamine-mediated signaling contributes to malaria pathogenesis. Understanding the basis for these biological effects of histamine during infection may lead to novel therapeutic strategies to alleviate the severity of malaria.

Horizontal eye movement networks in primates as revealed by retrograde transneuronal transfer of rabies virus: differences in monosynaptic input to "slow" and "fast" abducens motoneurons.

The sources of monosynaptic input to "fast" and "slow" abducens motoneurons (MNs) were revealed in primates by retrograde transneuronal tracing with rabies virus after injection either into the distal or central portions of the lateral rectus (LR) muscle, containing, respectively, "en grappe" endplates innervating slow muscle fibers or "en plaque" motor endplates innervating fast fibers. Rabies uptake involved exclusively motor endplates within the injected portion of the muscle. At 2.5 days after injections, remarkable differences of innervation of slow and fast MNs were demonstrated. Premotor connectivity of slow MNs, revealed here for the first time, involves mainly the supraoculomotor area, central mesencephalic reticular formation, and portions of medial vestibular and prepositus hypoglossi nuclei carrying eye position and smooth pursuit signals. Results suggest that slow MNs are involved exclusively in slow eye movements (vergence and possibly smooth pursuit), muscle length stabilization and gaze holding (fixation), and rule out their participation in fast eye movements (saccades, vestibulo-ocular reflex). By contrast, all known monosynaptic pathways to LR MNs innervate fast MNs, showing their participation in the entire horizontal eye movements repertoire. Hitherto unknown monosynaptic connections were also revealed, such as those derived from the central mesencephalic reticular formation and vertical eye movements pathways (Y group, interstitial nucleus of Cajal, rostral interstitial nucleus of the medial longitudinal fasciculus). The different connectivity of fast and slow MNs parallel differences in properties of muscle fibers that they innervate, suggesting that muscle fibers properties, rather than being self-determined, are the result of differences of their premotor innervation.

Density gradients of trans-synaptically labeled collicular neurons after injections of rabies virus in the lateral rectus muscle of the rhesus monkey.

We evaluated the two-dimensional distribution of superior colliculus (SC) neurons visualized after retrograde transneuronal transport of rabies virus injected into the lateral rectus muscle of rhesus monkeys to test whether the density of projection neurons might play a role in the spatiotemporal transformation and vector decomposition. If this were the case, the number of horizontal eye movement-related SC neurons should increase with their distance from the rostral pole of the SC and decrease with their distance from the representation of the horizontal meridian. Labeled neurons of the intermediate SC layers were counted inside a 1-mm-wide band that matched the horizontal meridian of the collicular motor map. Local areal densities were plotted against distance from the rostral SC pole. At 2.5 days after inoculation, there was no labeling in the SC. At 3 days, moderate labeling appeared on both sides, mostly in the intermediate layers. At 3.5 days, cell numbers substantially increased and the laminar distribution changed as cells appeared in the superficial SC layers. At 3 days, rostrocaudal density profiles were unimodal, with peaks at locations near 50 degrees (contralateral SC) and 25-30 degrees (ipsilateral SC) horizontal eccentricity. At 3.5 days, distributions were bimodal due to the appearance of a second high-density region near the rostral pole of the SC. The distribution of SC neurons influencing the abducens nucleus, thus, was nonuniform. Caudal sites contained more neurons, but the experimentally observed density gradients were shallower than the theoretically predicted ones that would be necessary to fully account for the spatiotemporal transformation. Similarly, we studied the distributions of cell densities in the intermediate SC layers along an isoamplitude line (representing saccades of equal amplitudes but different directions). Consistent with theoretical estimates of the density gradients required for vector decomposition, we found that the concentrations of labeled cells were highest in the vicinity of the horizontal meridian but their decrease toward the periphery of the motor map was steeper than predicted. We conclude that SC cell density gradients cannot fully account for the spatiotemporal transformation and vector decomposition in the absence of an additional mechanism such as the previously demonstrated (Grantyn et al., [1997] Soc. Neurosci. Abstr. 23:1295; Moschovakis et al., [1998] J. Neurosci. 18:10219-10229) locus-dependent weighting of the strength of efferent projections to the saccade generators.