Peroxisome proliferator-activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse.

The peroxisome proliferator-activated receptor gamma (PPARgamma) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPARgamma functions in mature adipocytes is precluded by lethality of PPARgamma(-/-) fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPARgamma in adipocytes of adult mice by using the tamoxifen-dependent Cre-ER(T2) recombination system. We show that mature PPARgamma-null white and brown adipocytes die within a few days and are replaced by newly formed PPARgamma-positive adipocytes, demonstrating that PPARgamma is essential for the in vivo survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPARgamma antagonists could be used to acutely reduce obesity.

Circadian clock-dependent and -independent rhythmic proteomes implement distinct diurnal functions in mouse liver.

Diurnal oscillations of gene expression controlled by the circadian clock underlie rhythmic physiology across most living organisms. Although such rhythms have been extensively studied at the level of transcription and mRNA accumulation, little is known about the accumulation patterns of proteins. Here, we quantified temporal profiles in the murine hepatic proteome under physiological light-dark conditions using stable isotope labeling by amino acids quantitative MS. Our analysis identified over 5,000 proteins, of which several hundred showed robust diurnal oscillations with peak phases enriched in the morning and during the night and related to core hepatic physiological functions. Combined mathematical modeling of temporal protein and mRNA profiles indicated that proteins accumulate with reduced amplitudes and significant delays, consistent with protein half-life data. Moreover, a group comprising about one-half of the rhythmic proteins showed no corresponding rhythmic mRNAs, indicating significant translational or posttranslational diurnal control. Such rhythms were highly enriched in secreted proteins accumulating tightly during the night. Also, these rhythms persisted in clock-deficient animals subjected to rhythmic feeding, suggesting that food-related entrainment signals influence rhythms in circulating plasma factors.

Evolution of the neurochemical profile after transient focal cerebral ischemia in the mouse brain.

Evolution of the neurochemical profile consisting of 19 metabolites after 30 mins of middle cerebral artery occlusion was longitudinally assessed at 3, 8 and 24 h in 6 to 8 microL volumes in the striatum using localized 1H-magnetic resonance spectroscopy at 14.1 T. Profound changes were detected as early as 3 h after ischemia, which include elevated lactate levels in the presence of significant glucose concentrations, decreases in glutamate and a transient twofold glutamine increase, likely to be linked to the excitotoxic release of glutamate and conversion into glial glutamine. Interestingly, decreases in N-acetyl-aspartate (NAA), as well as in taurine, exceeded those in neuronal glutamate, suggesting that the putative neuronal marker NAA is rather a sensitive marker of neuronal viability. With further ischemia evolution, additional, more profound concentration decreases were detected, reflecting a disruption of cellular functions. We conclude that early changes in markers of energy metabolism, glutamate excitotoxicity and neuronal viability can be detected with high precision non-invasively in mice after stroke. Such investigations should lead to a better understanding and insight into the sequential early changes in the brain parenchyma after ischemia, which could be used for identifying new targets for neuroprotection.

IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8(+) T cells.

CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.

Influence of triamcinolone intravitreal injection on retinochoroidal healing processes.

To analyze the effects of triamcinolone intravitreal injection on the wound healing processes after argon laser retinal photocoagulation, wild type C57BL/6J mice, 8-12 weeks old underwent a standard argon laser photocoagulation protocol. After pentobarbital anesthesia and pupil dilatation, argon laser lesions were induced (50microm, 400mW, 0.05s). Two photocoagulation impacts created two disc diameters from the optic nerve in both eyes. The photocoagulated mice were divided into four groups: Group I (n=12), photocoagulation controls, did not receive any intravitreous injection. Group II (n=12), received an intravitreous injection of 1microl of balanced salt solution (BSS). Group III (n=12), received an intravitreous injection of 1microl containing 15microg of triamcinolone acetonide (TAAC) in BSS. Two mice from each of these three groups were sacrificed at 1, 3, 7, 14 days and 2 and 4 months after photocoagulation. Group IV (n=10) received 1.5, 3, 7.5, 15, or 30microg of TAAC and were all sacrificed on day 14. The enucleated eyes were subjected to systematic analysis of the cellular remodeling processes taking place within the laser lesion and its vicinity. To this purpose, specific antibodies against GFAP, von Willebrand factor, F4/80 and KI67 were used for the detection of astrocytes, activated Müller cells, vascular endothelial cells, infiltrating inflammatory cells and actively proliferating cells. TUNEL reaction was also carried out along with nuclear DAPI staining. Temporal and spatial observations of the created photocoagulation lesions demonstrate that 24h following the argon laser beam, a localized and well-delineated affection of the RPE cells and choroid is observed in mice in Groups I and II. The inner retinal layers in these mice eyes are preserved while TUNEL positive (apoptotic) cells are observed at the retinal outer nuclear layer level. At this stage, intense staining with GFAP is associated with activated retinal astrocytes and Müller cells throughout the laser path. From day 3 after photocoagulation, dilated new choroidal capillaries are detected on the edges of the laser lesion. These processes are accompanied by infiltration of inflammatory cells and the presence of proliferating cells within the lesion site. Mice in Group III treated with 15microg/mul of triamcinolone showed a decreased number of infiltrating inflammatory cells and proliferating cells, which was not statistically significant compared to uninjected laser treated controls. The development of new choroidal capillaries on the edges of the laser lesion was also inhibited during the first 2 months after photocoagulation. However, on month 4 the growth of new vessels was observed in these mice treated with TAAC. Mice of Group IV did not show any development of new capillaries even with small doses. After argon laser photocoagulation of the mouse eye, intravitreal injection of triamcinolone markedly influenced the retina and choroid remodeling and healing processes. Triamcinolone is a powerful inhibitor of the formation of neovessels in this model. However, this inhibition is transient. These observations should provide a practical insight for the mode of TAAC use in patients with wet AMD.

T cell receptor V beta repertoire in mice lacking endogenous mouse mammary tumor provirus.

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) V beta segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, -7, -8 and -9) and 38CH (Mtv-) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, -7, -8 or -9. The TcR V beta chain (TcR V beta) usage in these mice was analyzed using monoclonal antibodies specific for TcR V beta 2, V beta 3, V beta 4, V beta 5, V beta 6, V beta 7, V beta 8, V beta 11, V beta 12 and V beta 14 segments. Both Mtv-8+ mice and Mtv-9+ mice deleted TcR V beta 5+ and V beta 11+ T cells. Moreover, we also observed the deletion of TcR V beta 12+ cells by Mtv-8 and Mtv-9 products. Mtv-6+ and Mtv-7+ animals deleted TcR V beta 3+ and V beta 5+ cells, and TcR V beta 6+, V beta 7+ and V beta 8.1+ cells, respectively. Unexpectedly, TcR V beta 8.2+ cells were also deleted in some backcross mice expressing Mtv-7. TcR V beta 8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn-->Asp) in position 19 on the TcR V beta 8.2 fragment. Reactivities of BALB.D2 TcR V beta 8.2 and 38CH TcR V beta 8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR V beta 2+, V beta 4+ and V beta 8+ CD4+ T cell subsets in Mtv-8+ and Mtv-9+ mice, and TcR V beta 4+ CD4+ T cells in Mtv-6+ and Mtv-7+ mice, when compared with the T cell repertoire of Mtv- mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.

Polyene macrolide antifungal drugs trigger interleukin-1β secretion by activating the NLRP3 inflammasome.

The use of antimycotic drugs in fungal infections is based on the concept that they suppress fungal growth by a direct killing effect. However, amphotericin and nystatin have been reported to also trigger interleukin-1β (IL-1β) secretion in monocytes but the molecular mechanism is unknown. Here we report that only the polyene macrolides amphotericin B, nystatin, and natamycin but none of the tested azole antimycotic drugs induce significant IL-1β secretion in-vitro in dendritic cells isolated from C57BL/6 mouse bone marrow. IL-1β release depended on Toll-like receptor-mediated induction of pro-IL-1β as well as the NLRP3 inflammasome, its adaptor ASC, and caspase-1 for enzymatic cleavage of pro-IL-1β into its mature form. All three drugs induced potassium efflux from the cells as a known mechanism for NLRP3 activation but the P2X7 receptor was not required for this process. Natamycin-induced IL-1β secretion also involved phagocytosis, as cathepsin activation as described for crystal-induced IL-1β release. Together, the polyene macrolides amphotericin B, nystatin, and natamycin trigger IL-1β secretion by causing potassium efflux from which activates the NLRP3-ASC-caspase-1. We conclude that beyond their effects on fungal growth, these antifungal drugs directly activate the host's innate immunity.

Role of neutrophils in "Leishmania" infection

Résumé : Dans le modèle murin d'infection avec le parasite protozoaire Leishmania major (L. major), la souche de souris C57BL/6 est résistante a |'infection et développe une réponse protectrice Thelper (Th) 1. Inversement, les souris de la souche BALB/c développent une réponse Th2 et sont sensibles a cette infection. A la suite d'une infection avec ce parasite, les neutrophiles sont les premières cellules présentes au site d'infection et sont recrutées de manière égale dans les souches résistantes et sensibles à L. major, Néanmoins, trois jours après l'infection, la majorité des neutrophiles disparaissent du site d'infection chez les souris C57BL/6, tandis que ils restent jusqu'a dix jours chez les souris BALB/c. Un rôle crucial des neutrophiles a été démontré durant l'infection avec L. major. En effet, la déplétion de ces cellules avant |'infection dans les souris BALB/c, conduit a une réduction du développement des lésions, associée à une baisse de la charge parasitaire et a une modification de la réponse immunitaire vers une réponse Th1 dans des souris normalement sensibles a |'infection, suggérant un rôle immunorégulateur de ces neutrophiles durant les premiers jours de l'infection. Dans la première partie de cette thèse, nous avons étudié le rôle des neutrophiles suite à l'infection avec L. major. Nous avons démontré que le parasite induisait des phénotypes de neutrophiles distincts chez les souris résistantes ou sensibles à L. major. Suite à l'exposition au parasite, les neutrophiles de souris C57BL/6 ont montré une expression élevée des récepteurs Toll-like 2, 7 et 9 ainsi que la sécrétion d'lL-12p7O et d'lL-10, alors que ceux de souris BALB/c sécrétaient de l'IL-12p40 et du TGFB. Nous avons ensuite démontré qu'en réponse à L. major, au contraire des neutrophiles de BALB/c, les neutrophiles de souris résistantes C57BL/6, libéraient la chimiokine CCL3 attirant les cellules dendritiques. Le rôle crucial de cette chimiokine dans la migration de la première de vague de cellules dendritiques au site d'infection ainsi que son rôle dans le développement de la réponse immunitaire subséquente a été établi. Ces résultats démontrent que les neutrophiles, suite a |'infection avec le parasite L. major, créent un microenvironnement capable de déterminer le développement d'une réponse immunitaire spécifique a un antigène. Dans un second temps, nous nous sommes intéressés au rôle des neutrophiles suite a l'infection avec d'autres espèces de Leishmania: L, doriovani et L. mexicaria, agents responsables de leishmaniose viscérale et cutanée chronique respectivement. Un rôle crucial des neutrophiles a été démontré dans la réponse protectrice suite a l'infection avec L. donovani, l'absence de ces cellules amenant à une susceptibilité au parasite accrue, associée avec une induction préférentielle d'une réponse Th2. Inversement, la déplétion des neutrophiles lors de l'infection avec L. mexicaria aboutit a une résistance accrue, comme constaté par la baisse dela charge parasitaire, la hausse de la réponse Th1 ainsi la baisse de la réponse Th2 dans les souris déplétées en neutrophiles. Néanmoins, malgré le rôle délétère des neutrophiles sur le développement d'une réponse protectrice suite à |'infection avec L. mexicana, ces cellules sont nécessaires pour une résolution correcte dela réponse inflammatoire. En résumé, cette étude révèle un rôle majeur des neutrophiles lors de |'infection avec plusieurs especes de Leishmania. Résumé pour un large public : Les neutrophiles font partie de la famille des globules blancs. A la suite d'une infection, ces cellules sont les premières a être recrutées au site d'infection et sont impliquées dans |'élimination des pathogènes. Dans cette thèse, nous nous somme donc intéressés au rôle que pouvaient jouer ces neutrophiles durant l'infection avec le parasite protozoaire Leishmania major (L. major). Dans le modèle murin d'infection avec L. major, la majorité des souches de souris utilisées dans la recherche, dont les souris de la souche C57BL/6, développent de petites lésions qui guérissent spontanément après quelques semaines (souris résistantes). ll existe néanmoins, quelques souches de souris, dont la souche de souris BALB/c, qui développent des lésions qui ne guérissent pas (souris sensibles). Il a été observé que lors de l'lnfection avec ce parasites les neutrophiles étaient les premières cellules recrutées au site de l'lnfection dans toutes les souches de souris, toutefois trois jours après le début dela réaction immunitaire, la majorité des neutrophiles disparaissent chez les souris C57BL/6, tandis qu'ils restent jusqu'à dix jours chez les souris BALB/c. De plus, un rôle crucial des neutrophiles a été démontré durant l'infection avec L. major. En effet, l'absence de neutrophiles durant les trois premiers jours de l'infection chez les souris sensibles à |'infection, rend ces souris résistantes. Ces résultats suggèrent donc un rôle régulateur de la réponse immunitaire des neutrophiles durant les premiers jours de l'infection. Dans la première partie de cette thèse, nous avons étudié le rôle des neutrophiles suite à l'infection avec L. major. Nous avons donc analysé la sécrétion des cytokines, molécules essentielles qui déterminent la réponse immunitaire, par les neutrophiles. Nous avons démontré que le parasite induisait une sécrétion de cytokines différente entre les souris résistantes ou sensibles a L. major. Nous avons ensuite démontré que seule la souche de souris résistante sécrétait la chimiokine CCL3, connue pour être impliquée dans le recrutement de différentes cellules au site d'infecti0n, dont les cellules dendritiques. Les cellules dendritiques sont un élément fondamental pour un bon déroulement d'une réponse immunitaire, de par leur rôle décisif de liaison entre une réponse précoce non-spécifique au pathogène et une réponse plus tardive spécifique au pathogène et nécessaire pour |'élimination de dernier. Nous avons démontré que les neutrophiles de souris résistantes sécrétaient CCL3 et recrutaient les cellules dendritiques au site d'infecti0n, jouant de ce fait un rôle essentiel dans le développement de la réponse immunitaire. Ces résultats démontrent que les neutrophiles, suite à l'infection avec le parasite L. major, créent un microenvironnement capable de déterminer le développement d'une réponse immunitaire. Dans un second temps, nous nous sommes intéressés au rôle des neutrophiles suite à l'lnfection avec d'autres espèces de Leishmania, L. donovani et L. mexicana. Nous avons pu montrer un rôle crucial de ces cellules dans la réponse à ces deux parasites. En effet, suite à |'infection avec L. donovani, un rôle protecteur des neutrophiles a été observé, leur absence menant à une susceptibilité accrue aux parasites. Dans le cas de l'infection avec L. mexicana, une réduction de |'infection a été observée en absence de neutrophiles, avec néanmoins une augmentation de la lésion, suggérant un rôle important de ces cellules dans le développement de la réponse immunitaire ainsi que dans le contrôle de la réponse inflammatoire. En résumé, cette étude révèle un rôle majeur des neutrophiles lors de l'lnfection avec plusieurs membres de la famille Leishrnania. Summary : Upon infection with the protozoan parasite Leishmania major (L. major), C57BL/6 mice show a resistant phenotype, developing a protective Thelper (Th) 1 response. ln contrast, BALB/c mice develop a Th2 response and are susceptible to infection. Following inoculation with the parasite, neutrophils are the first cells migrating at the site of infection and are equally recruited in both L. major- resistant and susceptible mouse strains. However, after three days of infection, almost all neutrophils disappear from the site of infection in C57BL/6 mice, while they persist until ten days in BALB/c mice. Neutrophils were shown to play a crucial role during infection with L. major. indeed, depletion of these cells in BALB/c mice prior to infection with the parasite led to a lower Iesion development, associated with a lower parasite burden and a modification in the immune response towards a Th1 response in these otherwise susceptible mice, suggesting an immunomodulatory role for neutrophils during the first days of infection. ln the first part of this thesis, we were interested in better understanding the role of neutrophils in infection with L. major. \/\/e found that this parasite was inducing distinct neutrophil phenotypes in L. major-resistant and susceptible mice. Upon exposition with L. major, C57BL/6 neutrophils were reported to express high level of Toll-like receptors 2, 7, 9 mRNA and secrete IL-12p70 and IL-10, while BALB/c neutrophils secreted homodimers of IL-12p40, and TGFB. We then demonstrated that in response to L. major, neutrophils from L. major-resistant C57BL/6 mice release the CCL3 dendritic cell attracting chemokine, which is critical for the first wave of dendritic cell migration to the site of infection and in the development of the subsequent immune response. Altogether, these results demonstrated that upon infection with L. major, neutrophils create a microenvironment that can determine the development of an antigen-specific immune response. ln the second part of the thesis we were interested in understanding the role of neutrophils upon infection with of other species of Leishmania: L. donovani causing visceral leishmaniasis and L. mexicana, agent of chronic cutaneous leishmaniasis. Upon infection with L. donovani, neutrophils were found to play a crucial role in the early protective response, their absence leading to an increased susceptibility to the parasite, associated with the preferential induction of a Th2 response. ln contrast, depletion of these cells early in infection with L. mexicana was leading to an increased resistance, as observed by a decreased parasite burden, increased Th1 and decreased Th2 response in neutrophil-depleted mice. However, despite the deleterious role of neutrophils on the development of a protective immune response upon L. mexicana infection, these cells were required for the proper resolution of the inflammatory response. Altogether, these results highlight a major immunomodulatory role for neutrophils in infection with several species of Leishmania.

Distribution of neuropeptide Y Y1 receptors in rodent peripheral tissues.

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking alpha-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R.

An allele-specific, stochastic gene expression process controls the expression of multiple Ly49 family genes and generates a diverse, MHC-specific NK cell receptor repertoire.

Mouse NK cells express MHC class I-specific inhibitory Ly49 receptors. Since these receptors display distinct ligand specificities and are clonally distributed, their expression generates a diverse NK cell receptor repertoire specific for MHC class I molecules. We have previously found that the Dd (or Dk)-specific Ly49A receptor is usually expressed from a single allele. However, a small fraction of short-term NK cell clones expressed both Ly49A alleles, suggesting that the two Ly49A alleles are independently and randomly expressed. Here we show that the genes for two additional Ly49 receptors (Ly49C and Ly49G2) are also expressed in a (predominantly) mono-allelic fashion. Since single NK cells can co-express multiple Ly49 receptors, we also investigated whether mono-allelic expression from within the tightly linked Ly49 gene cluster is coordinate or independent. Our clonal analysis suggests that the expression of alleles of distinct Ly49 genes is not coordinate. Thus Ly49 alleles are apparently independently and randomly chosen for stable expression, a process that directly restricts the number of Ly49 receptors expressed per single NK cell. We propose that the Ly49 receptor repertoire specific for MHC class I is generated by an allele-specific, stochastic gene expression process that acts on the entire Ly49 gene cluster.

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

NR2A at CA1 synapses is obligatory for the susceptibility of hippocampal plasticity to sleep loss.

A loss in the necessary amount of sleep alters expression of genes and proteins implicated in brain plasticity, but key proteins that render neuronal circuits sensitive to sleep disturbance are unknown. We show that mild (4-6 h) sleep deprivation (SD) selectively augmented the number of NR2A subunits of NMDA receptors on postsynaptic densities of adult mouse CA1 synapses. The greater synaptic NR2A content facilitated induction of CA3-CA1 long-term depression in the theta frequency stimulation range and augmented the synaptic modification threshold. NR2A-knock-out mice maintained behavioral response to SD, including compensatory increase in post-deprivation resting time, but hippocampal synaptic plasticity was insensitive to sleep loss. After SD, the balance between synaptically activated and slowly recruited NMDA receptor pools during temporal summation was disrupted. Together, these results indicate that NR2A is obligatory for the consequences of sleep loss on hippocampal synaptic plasticity. These findings could advance pharmacological strategies aiming to sustain hippocampal function during sleep restriction.

Rufinamide Attenuates Mechanical Allodynia in a Model of Neuropathic Pain in the Mouse and Stabilizes Voltage-gated Sodium Channel Inactivated State.

BACKGROUND:: Voltage-gated sodium channels dysregulation is important for hyperexcitability leading to pain persistence. Sodium channel blockers currently used to treat neuropathic pain are poorly tolerated. Getting new molecules to clinical use is laborious. We here propose a drug already marketed as anticonvulsant, rufinamide. METHODS:: We compared the behavioral effect of rufinamide to amitriptyline using the Spared Nerve Injury neuropathic pain model in mice. We compared the effect of rufinamide on sodium currents using in vitro patch clamp in cells expressing the voltage-gated sodium channel Nav1.7 isoform and on dissociated dorsal root ganglion neurons to amitriptyline and mexiletine. RESULTS:: In naive mice, amitriptyline (20 mg/kg) increased withdrawal threshold to mechanical stimulation from 1.3 (0.6-1.9) (median [95% CI]) to 2.3 g (2.2-2.5) and latency of withdrawal to heat stimulation from 13.1 (10.4-15.5) to 30.0 s (21.8-31.9), whereas rufinamide had no effect. Rufinamide and amitriptyline alleviated injury-induced mechanical allodynia for 4 h (maximal effect: 0.10 ± 0.03 g (mean ± SD) to 1.99 ± 0.26 g for rufinamide and 0.25 ± 0.22 g to 1.92 ± 0.85 g for amitriptyline). All drugs reduced peak current and stabilized the inactivated state of voltage-gated sodium channel Nav1.7, with similar effects in dorsal root ganglion neurons. CONCLUSIONS:: At doses alleviating neuropathic pain, amitriptyline showed alteration of behavioral response possibly related to either alteration of basal pain sensitivity or sedative effect or both. Side-effects and drug tolerance/compliance are major problems with drugs such as amitriptyline. Rufinamide seems to have a better tolerability profile and could be a new alternative to explore for the treatment of neuropathic pain.

RNA interference mitigates motor and neuropathological deficits in a cerebellar mouse model of machado-joseph disease.

Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease.

Two-kidney, one clip and one-kidney, one clip hypertension in mice.

The mouse remains the animal of choice in transgenic experiments, creating a need for methods of evaluating the physiology of genetically modified animals. We have established and characterized two murine models of renovascular hypertension known as the two-kidney, one clip and one-kidney, one clip models. The appropriate size of the clip lumen needed to induce high blood pressure was determined to be 0.12 mm. Clips with a lumen of 0.11 mm induced a high percentage of renal infarction, and clips with a 0.13-mm opening did not produce hypertension. Four weeks after clipping, two-kidney, one clip hypertensive mice exhibited blood pressure approximately 20 mm Hg higher than their sham-operated controls. After a similar period, this increase reached almost 35 mm Hg in the one-kidney, one clip model. Depending on the model, mice develop either renin-dependent or renin-independent hypertension. Both models are characterized by the development of cardiovascular hypertrophy.