Homeostasis limits the development of mature CD8+ but not CD4+ thymocytes.

The involvement of a variety of clonal selection processes during the development of T lymphocytes in the thymus has been well established. Less information, however, is available on how homeostatic mechanisms may regulate the generation and maturation of thymocytes. To investigate this question, mixed radiation bone marrow chimeras were established in which wild-type T cell precursors capable of full maturation were diluted with precursors deficient in maturation potential because of targeted mutations of the RAG1 or TCR-alpha genes. In chimeras in which the majority of thymocytes are blocked at the CD4- CD8- CD25+ stage (RAG1 deficient), and only a small proportion of T cell precursors are of wild-type origin, we observed no difference in the maturation of wild-type CD4- CD8- CD25+ cells to the CD4+ CD8+ stage as compared with control chimeras. Therefore, the number of cell divisions occurring during this transition is fixed and not subject to homeostatic regulation. In contrast, in mixed chimeras in which the majority of thymocytes are blocked at the CD4+ CD8+ stage (TCR-alpha deficient), an increased efficiency of development of wild-type mature CD8+ cells was observed. Surprisingly, the rate of generation of mature CD4+ thymocytes was not affected in these chimeras. Thus, the number of selectable CD8 lineage thymocytes apparently saturates the selection mechanism in normal mice while the development of CD4 lineage cells seems to be limited only by the expression of a suitable TCR. These data may open the way to the identification of homeostatic mechanisms regulating thymic output and CD4/CD8 lineage commitment, and the development of means to modulate it.

On the origin of the MR image phase contrast: an in vivo MR microscopy study of the rat brain at 14.1 T.

Recent studies at high magnetic fields using the phase of gradient-echo MR images have shown the ability to unveil cortical substructure in the human brain. To investigate the contrast mechanisms in phase imaging, this study extends, for the first time, phase imaging to the rodent brain. Using a 14.1 T horizontal bore animal MRI scanner for in vivo micro-imaging, images with an in-plane resolution of 33 microm were acquired. Phase images revealed, often more clearly than the corresponding magnitude images, hippocampal fields, cortical layers (e.g. layer 4), cerebellar layers (molecular and granule cell layers) and small white matter structures present in the striatum and septal nucleus. The contrast of the phase images depended in part on the orientation of anatomical structures relative to the magnetic field, consistent with bulk susceptibility variations between tissues. This was found not only for vessels, but also for white matter structures, such as the anterior commissure, and cortical layers in the cerebellum. Such susceptibility changes could result from variable blood volume. However, when the deoxyhemoglobin content was reduced by increasing cerebral blood flow (CBF) with a carbogen breathing challenge, contrast between white and gray matter and cortical layers was not affected, suggesting that tissue cerebral blood volume (and therefore deoxyhemoglobin) is not a major source of the tissue phase contrast. We conclude that phase variations in gradient-echo images are likely due to susceptibility shifts of non-vascular origin.

The value of serum procalcitonin level for differentiation of infectious from noninfectious causes of fever after orthopaedic surgery.

BACKGROUND: Early diagnosis of postoperative orthopaedic infections is important in order to rapidly initiate adequate antimicrobial therapy. There are currently no reliable diagnostic markers to differentiate infectious from noninfectious causes of postoperative fever. We investigated the value of the serum procalcitonin level in febrile patients after orthopaedic surgery. METHODS: We prospectively evaluated 103 consecutive patients with new onset of fever within ten days after orthopaedic surgery. Fever episodes were classified by two independent investigators who were blinded to procalcitonin results as infectious or noninfectious origin. White blood-cell count, C-reactive protein level, and procalcitonin level were assessed on days 0, 1, and 3 of the postoperative fever. RESULTS: Infection was diagnosed in forty-five (44%) of 103 patients and involved the respiratory tract (eighteen patients), urinary tract (eighteen), joints (four), surgical site (two), bloodstream (two), and soft tissues (one). Unlike C-reactive protein levels and white blood-cell counts, procalcitonin values were significantly higher in patients with infection compared with patients without infection on the day of fever onset (p = 0.04), day 1 (p = 0.07), and day 3 (p = 0.003). Receiver-operating characteristics demonstrated that procalcitonin had the highest diagnostic accuracy, with a value of 0.62, 0.62, and 0.71 on days 0, 1, and 3, respectively. In a multivariate logistic regression analysis, procalcitonin was a significant predictor for postoperative infection on days 0, 1, and 3 of fever with an odds ratio of 2.3 (95% confidence interval, 1.1 to 4.4), 2.3 (95% confidence interval, 1.1 to 5.2), and 3.3 (95% confidence interval, 1.2 to 9.0), respectively. CONCLUSIONS: Serum procalcitonin is a helpful diagnostic marker supporting clinical and microbiological findings for more reliable differentiation of infectious from noninfectious causes of fever after orthopaedic surgery.

Epidermal growth factor (EGF) stimulation of cultured brain cells. II. Increased production of extracellular soluble proteins.

The production of extracellular soluble proteins was studied in serum-free aggregating cell cultures of fetal rat telencephalon labeled on culture day 7 with a mixture of radioactive amino acid precursors. Cultures treated continuously with epidermal growth factor (EGF; 20 ng/ml) showed a generally increased protein secretion and a particularly enhanced production of a few distinct extracellular proteins. The time lag of this response after an initial dose of EGF (25 ng/ml) on day 7 was 48 h. The total macromolecular radioactivity that accumulated within 96 h of labeling in the media of EGF-treated cultures was 175% of untreated controls, whereas no difference was found in the proportions of intracellular amino acid incorporation. Cultures which received a single dose of EGF (25 ng/ml) on day 1 showed still a greatly increased protein secretion on day 7. Prevention of extracellular protein accumulation by reducing the initial cell number and increasing the rate of media changes did not affect the EGF-induced stimulation of the two glial enzymes, glutamine synthetase and 2',3'-cyclic nucleotide 3'-phosphohydrolase. The results suggest that both the increased production of extracellular proteins and the enhanced development of glial enzymatic activities reflect the stimulated phenotypic expression of EGF-sensitive brain cells.

Effects of lactate on glucose metabolism in healthy subjects and in severely injured hyperglycemic patients.

Hepatic glucose production is autoregulated during infusion of gluconeogenic precursors. In hyperglycemic patients with multiple trauma, hepatic glucose production and gluconeogenesis are increased, suggesting that autoregulation of hepatic glucose production may be defective. To better understand the mechanisms of autoregulation and its possible alterations in metabolic stress, lactate was coinfused with glucose in healthy volunteers and in hyperglycemic patients with multiple trauma or critical illness. In healthy volunteers, infusion of glucose alone nearly abolished endogenous glucose production. Lactate increased gluconeogenesis (as indicated by a decrease in net carbohydrate oxidation with no change in total [13C]carbohydrate oxidation) but did not increase endogenous glucose production. In patients with metabolic stress, endogenous glucose production was not suppressed by exogenous glucose, but lactate did not further increase hepatic glucose production. It is concluded that 1) in healthy humans, autoregulation of hepatic glucose production during infusion of lactate is still present when glycogenolysis is suppressed by exogenous glucose and 2) autoregulation of hepatic glucose production is not abolished in hyperglycemic patients with metabolic stress.

Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate.

Glucagon-like peptide-1 (GLP-1) stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor linked to activation of the adenylyl cyclase pathway. Here, using insulinoma cell lines, we studied homologous and heterologous desensitization of GLP-1-induced cAMP production. Preexposure of the cells to GLP-1 induced a decrease in GLP-1-mediated cAMP production, as assessed by a 3- to 5-fold rightward shift of the dose-response curve and an approximately 20 percent decrease in the maximal production of cAMP. Activation of protein kinase C by the phorbol ester phorbol 12-myristate 13-acetate (PMA) also induced desensitization of the GLP-1-mediated response, leading to a 6- to 9-fold shift in the EC50 and a 30% decrease in the maximal production of cAMP. Both forms of desensitization were additive, and the protein kinase C inhibitor RO-318220 inhibited PMA-induced desensitization, but not agonist-induced desensitization. GLP-1- and PMA-dependent desensitization correlated with receptor phosphorylation, and the levels of phosphorylation induced by the two agents were additive. Furthermore, PMA-induced, but not GLP-1-induced, phosphorylation was totally inhibited by RO-318220. Internalization of the GLP-1 receptor did not participate in the desensitization induced by PMA, as a mutant GLP-1 receptor lacking the last 20 amino acids of the cytoplasmic tail was found to be totally resistant to the internalization process, but was still desensitized after PMA preexposure. PMA and GLP-1 were not able to induce the phosphorylation of a receptor deletion mutant lacking the last 33 amino acids of the cytoplasmic tail, indicating that the phosphorylation sites were located within the deleted region. The cAMP production mediated by this deletion mutant was not desensitized by PMA and was only poorly desensitized by GLP-1. Together, our results indicate that the production of cAMP and, hence, the stimulation of insulin secretion induced by GLP-1 can be negatively modulated by homologous and heterologous desensitization, mechanisms that involve receptor phosphorylation.

Cerebral extracellular lactate increase is predominantly non ischemic in patients with severe traumatic brain injury

Le cerveau est l'organe avec les besoins en énergie les plus élevés du corps humain, et le glucose est un substrat énergétique cérébral essentiel. Ces dernières décennies, la compréhension de la neuroénergétique a beaucoup évolué et un rôle du lactate comme substrat énergétique important a été mis en évidence, notamment suite à l'introduction du modèle de l'ANLS (astrocyte-neuron lactate shuttle). Selon celui-ci, les astrocytes convertissent le glucose en lactate par réaction de glycolyse, puis il est transporté jusqu'aux neurones qui l'utilisent comme source d'énergie à travers le cycle de Krebs. Chez l'homme, divers travaux récents ont montré que le lactate peut servir de « carburant » cérébral chez le sujet sain, après effort intense ou chez le patient diabétique. La régulation métabolique et le rôle du lactate après lésion cérébrale aiguë sont encore peu connus. Présentation de l'article Le but de ce travail a été d'étudier le métabolisme cérébral du lactate chez les patients atteints de traumatisme crânien (TCC) sévère. Nous avons émis l'hypothèse que l'augmentation du lactate cérébral chez ces patients n'était pas associée de manière prédominante à une hypoxie ou une ischémie mais plutôt à une glycolyse aérobie, et également à une perfusion cérébrale normale. L'étude a porté sur une cohorte prospective de 24 patients avec TCC sévère admis au service de médecine intensive du CHUV (centre hospitalier universitaire vaudois), monitorés par un système combinant microdialyse cérébrale (outil permettant de mesurer divers métabolites cérébraux, tels que le lactate, le pyruvate et le glucose), mesure de la pression cérébrale en oxygène et de la pression intracrânienne. Cet outil nous a permis de déterminer si l'élévation du lactate était principalement associée à une glycolyse active ou plutôt à une hypoxie. L'utilisation du CTde perfusion a permis d'évaluer la relation entre les deux patterns d'élévation du lactate (glycolytique ou hypoxique) et la perfusion cérébrale globale. Nos résultats ont montré que l'augmentation du lactate cérébral chez les patients avec TCC sévère était associée de manière prédominante à une glycolyse aérobie plutôt qu'à une hypoxie/ischémie. D'autre part, nous avons pu confirmer que les épisodes de lactate glycolytique étaient toujours associés à une perfusion cérébrale normale ou augmentée, alors que les épisodes de lactate hypoxique étaient associés à une hypoperfusion cérébrale. Conclusions et perspectives Nos résultats, qui ont permis de mieux comprendre le métabolisme cérébral du lactate chez les patients avec TCC sévère, soutiennent le concept que le lactate est produit dans des conditions aérobes et pourrait donc être utilisé comme source d'énergie par le cerveau lésé pour subvenir à des besoins augmentas. Etant donné que la dysfonction énergétique est une des probables causes de perte neuronale après traumatisme crânien, ces résultats ouvrent des perspectives thérapeutiques nouvelles après agression cérébrale chez l'homme, visant à tester un potentiel effet neuroprotecteur via l'administration de lactate exogène.

Gliotransmission in the hippocampus, mechanisms and interaction with synaptic functions

SUMMARYAstrocytes represent the largest cell population in the human brain. In addition to a well established role as metabolic support for neuronal activity, in the last years these cells have been found to accomplish other important and, sometimes, unexpected functions. The tight enwrapping of synapses by astrocytic processes and the predominant expression of glutamate uptake carriers in the astrocytic rather than neuronal plasma membranes brought to the definition of a critical involvement of astrocytes in the clearance of glutamate from synaptic junctions. Moreover, several publications showed that astrocytes are able to release chemical transmitters (gliotransmitters) suggesting their active implication in the control of synaptic functions. Among gliotransmitters, the best characterized is glutamate, which has been proposed to be released from astrocytes in a Ca2+ dependent manner via exocytosis of synaptic-like microvesicles.In my thesis I present results leading to substantial advancement of the understanding of the mechanisms by which astrocytes modulate synaptic activity in the hippocampus, notably at excitatory synapses on dentate granule cells. I show that tumor necrosis factor- alpha (TNFa), a molecule that is generally involved in immune system functions, critically controls astrocyte-to-synapse communication (gliotransmission) in the brain. With constitutive levels of TNFa present, activation of purinergic G protein-coupled receptors in astrocytes, called P2Y1 receptors, induces localized intracellular calcium ([Ca2+]j) elevation in astrocytic processes (measured by two-photon microscopy) followed by glutamate release and activation of pre-synaptic NMDA receptors resulting in synaptic potentiation. In preparations lacking TNFa, astrocytes respond with identical [Ca2+]i elevations but fail to induce neuromodulation. I find that TNFa specifically controls the glutamate release step of gliotransmission. Addition of very low (picomolar) TNFa concentrations to preparations lacking the cytokine, promptly reconstitutes both normal exocytosis in cultured astrocytes and gliotransmission in hippocampal slices. These data provide the first demonstration that gliotransmission and its synaptic effects are controlled not only by astrocyte [Ca2+]i elevations but also by permissive/homeostatic factors like TNFa.In addition, I find that higher and presumably pathological TNFa concentrations do not act just permissively but instead become direct and potent triggers of glutamate release from astrocytes, leading to a strong enhancement of excitatory synaptic activity. The TNFa action, like the one observed upon P2Y1R activation, is mediated by pre-synaptic NMDA receptors, but in this case the effect is long-lasting, and not reversible. Moreover, I report that a necessary molecular target for this action of TNFa is TNFR1, one of the two specific receptors for the cytokine, as I found that TNFa was unable to induce synaptic potentiation when applied in slices from TNFR1 knock-out (Tnfrlv") mice. I then created a double transgenic mouse model where TNFR1 is knocked out in all cells but can be re-expressed selectively in astrocytes and I report that activation of the receptors in these cells is sufficient to reestablish TNFa-dependent long-lasting potentiation of synaptic activity in the TNFR1 knock-out mice.I therefore discovered that TNFa is a primary molecule displaying both permissive and instructive roles on gliotransmission controlling synaptic functions. These reports might have profound implications for the understanding of both physiological and pathological processes associated to TNFa production, including inflammatory processes in the brain.RÉSUMÉLes astrocytes sont les cellules les plus abondantes du cerveau humain. Outre leur rôle bien établi dans le support métabolique de l'activité neuronale, d'autres fonctions importantes, et parfois inattendues de ces cellules ont été mises en lumière au cours de ces dernières années. Les astrocytes entourent étroitement les synapses de leurs fins processus qui expriment fortement les transporteurs du glutamate et permettent ainsi aux astrocytes de jouer un rôle critique dans l'élimination du glutamate de la fente synaptique. Néanmoins, les astrocytes semblent être capables de jouer un rôle plus intégratif en modulant l'activité synaptique, notamment par la libération de transmetteurs (gliotransmetteurs). Le gliotransmetteur le plus étudié est le glutamate qui est libéré par l'exocytose régulée de petites vésicules ressemblant aux vésicules synaptiques (SLMVs) via un mécanisme dépendant du calcium.Les résultats présentés dans cette thèse permettent une avancée significative dans la compréhension du mode de communication de ces cellules et de leur implication dans la transmission de l'information synaptique dans l'hippocampe, notamment des synapses excitatrices des cellules granulaires du gyrus dentelé. J'ai pu montrer que le « facteur de nécrose tumorale alpha » (TNFa), une cytokine communément associée au système immunitaire, est aussi fondamentale pour la communication entre astrocyte et synapse. Lorsqu'un niveau constitutif très bas de TNFa est présent, l'activation des récepteurs purinergiques P2Y1 (des récepteurs couplés à protéine G) produit une augmentation locale de calcium (mesurée en microscopie bi-photonique) dans l'astrocyte. Cette dernière déclenche ensuite une libération de glutamate par les astrocytes conduisant à l'activation de récepteurs NMDA présynaptiques et à une augmentation de l'activité synaptique. En revanche, dans la souris TNFa knock-out cette modulation de l'activité synaptique par les astrocytes n'est pas bien qu'ils présentent toujours une excitabilité calcique normale. Nous avons démontré que le TNFa contrôle spécifiquement l'exocytose régulée des SLMVs astrocytaires en permettant la fusion synchrone de ces vésicules et la libération de glutamate à destination des récepteurs neuronaux. Ainsi, nous avons, pour la première fois, prouvé que la modulation de l'activité synaptique par l'astrocyte nécessite, pour fonctionner correctement, des facteurs « permissifs » comme le TNFa, agissant sur le mode de sécrétion du glutamate astrocytaire.J'ai pu, en outre, démontrer que le TNFa, à des concentrations plus élevées (celles que l'on peut observer lors de conditions pathologiques) provoque une très forte augmentation de l'activité synaptique, agissant non plus comme simple facteur permissif mais bien comme déclencheur de la gliotransmission. Le TNFa provoque 1'activation des récepteurs NMD A pré-synaptiques (comme dans le cas des P2Y1R) mais son effet est à long terme et irréversible. J'ai découvert que le TNFa active le récepteur TNFR1, un des deux récepteurs spécifiques pour le TNFa. Ainsi, l'application de cette cytokine sur une tranche de cerveau de souris TNFR1 knock-out ne produit aucune modification de l'activité synaptique. Pour vérifier l'implication des astrocytes dans ce processus, j'ai ensuite mis au point un modèle animal doublement transgénique qui exprime le TNFR1 uniquement dans les astrocytes. Ce dernier m'a permis de prouver que l'activation des récepteurs TNFR1 astrocytaires est suffisante pour induire une augmentation de l'activité synaptique de manière durable.Nous avons donc découvert que le TNFa possède un double rôle, à la fois un rôle permissif et actif, dans le contrôle de la gliotransmission et, par conséquent, dans la modulation de l'activité synaptique. Cette découverte peut potentiellement être d'une extrême importance pour la compréhension des mécanismes physiologiques et pathologiques associés à la production du TNFa, en particulier lors de conditions inflammatoires.RÉSUMÉ GRAND PUBLICLes astrocytes représentent la population la plus nombreuse de cellules dans le cerveau humain. On sait, néanmoins, très peu de choses sur leurs fonctions. Pendant très longtemps, les astrocytes ont uniquement été considérés comme la colle du cerveau, un substrat inerte permettant seulement de lier les cellules neuronales entre elles. Il n'y a que depuis peu que l'on a découvert de nouvelles implications de ces cellules dans le fonctionnement cérébral, comme, entre autres, une fonction de support métabolique de l'activité neuronale et un rôle dans la modulation de la neurotransmission. C'est ce dernier aspect qui fait l'objet de mon projet de thèse.Nous avons découvert que l'activité des synapses (régions qui permettent la communication d'un neurone à un autre) qui peut être potentialisée par la libération du glutamate par les astrocytes, ne peut l'être que dans des conditions astrocytaires très particulières. Nous avons, en particulier, identifié une molécule, le facteur de nécrose tumorale alpha (TNFa) qui joue un rôle critique dans cette libération de glutamate astrocytaire.Le TNFa est surtout connu pour son rôle dans le système immunitaire et le fait qu'il est massivement libéré lors de processus inflammatoires. Nous avons découvert qu'en concentration minime, correspondant à sa concentration basale, le TNFa peut néanmoins exercer un rôle indispensable en permettant la communication entre l'astrocyte et le neurone. Ce mode de fonctionnement est assez probablement représentatif d'un processus physiologique qui permet d'intégrer la communication astrocyte/neurone au fonctionnement général du cerveau. Par ailleurs, nous avons également démontré qu'en quantité plus importante, le TNFa change son mode de fonctionnement et agit comme un stimulateur direct de la libération de glutamate par l'astrocyte et induit une activation persistante de l'activité synaptique. Ce mode de fonctionnement est assez probablement représentatif d'un processus pathologique.Nous sommes également arrivés à ces conclusions grâce à la mise en place d'une nouvelle souche de souris doublement transgéniques dans lesquelles seuls les astrocytes (etnon les neurones ou les autres cellules cérébrales) sont capables d'être activés par le TNFa.

Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells.

Thymic dendritic cells (DCs) form a discrete subset of bone marrow (BM)-derived cells, the function of which is to mediate negative selection of autoreactive thymocytes. The developmental origin of thymic DCs remains controversial. Although cell transfer studies support a model in which T cells and thymic DCs develop from the same intrathymic pluripotential precursor, it remains possible that these two types of cells develop from independent intrathymic precursors. Notch proteins are cell surface receptors involved in the regulation of cell fate specification. We have recently reported that T cell development in inducible Notch1-deficient mice is severely impaired at an early stage, before the expression of T cell lineage markers. To investigate whether development of thymic DCs also depends on Notch1, we have constructed mixed BM chimeric mice. We report here that thymic DC development from Notch1(-/)- BM precursors is absolutely normal (in terms of absolute number and phenotype) in this competitive situation, despite the absence of Notch1(-/)- T cells. Furthermore, we find that peripheral DCs and Langerhans cells are also not affected by Notch1 deficiency. Our results demonstrate that the development of DCs is totally independent of Notch1 function, and strongly suggest a dissociation between intrathymic T cell and DC precursors.

Brain energetics (thought needs food).

PURPOSE OF REVIEW: Almost 15 years after its initial proposal, the astrocyte-neuron lactate shuttle hypothesis still occupies the center stage in research on brain energetics. Recent developments have provided further evidence for its validity and have extended its application to different areas of neuroscience. RECENT FINDINGS: Description of cell-specific metabolic characteristics have reinforced the view that a prominent conversion of glucose into lactate takes place in astrocytes, whereas neurons preferentially take up and oxidize lactate over glucose-derived pyruvate. Indeed, specific mechanisms are activated by glutamatergic activity to favor such a net lactate transfer between the two cell types. Moreover, demonstration in vivo of the existence and implication of the astrocyte-neuron lactate shuttle hypothesis for particular neurophysiological processes is beginning to appear. SUMMARY: Brain energetics has undertaken its revolution. A new concept based on metabolic compartmentalization between astrocytes and neurons is establishing itself as the leading paradigm that opens new perspectives in areas such as functional brain imaging and regulation of energy homeostasis.

Distribution of neuronal and metabolic markers in the human auditory cortex

SUMMARY The human auditory cortex, located on the supratemporal plane of the temporal lobe, is divided in a primary auditory area and several non-primary areas surrounding it. These different areas show anatomical and functional differences. Many studies have focussed on auditory areas in non-human primates, using investigation techniques such as electrophysiological recordings, tracing of neural connections, or immunohistochemical and histochemical staining. Some of these studies have suggested parallel and hierarchical organization of the cortical auditory areas as well as subcortical auditory relays. In humans, only few studies have investigated these regions immunohistochemically, but activation and lesion studies speak in favour of parallel and hierarchical organization, very similar to that of non-human primates. Calcium-binding proteins and metabolic markers were used to investigate possible correlates of hierarchical and parallel organization in man. Calcium-binding proteins, parvalbumin, calretinin and calbindin, modulate the concentration of intracellular free calcium ions and were found in distinct subpopulations of GABAergic neurons in non-human primates species. In our study, their distribution showed several differences between auditory areas: the primary auditory area was darkly stained for both parvalbumin and calbindin, and their expression rapidly decreased while moving away from the primary area. This staining pattern suggests a hierarchical organization of the areas, in which the more darkly stained areas could correspond to an earlier integration level and the areas showing light staining may correspond to higher level integration areas. Parallel organization of primary and non-primary auditory areas was suggested by the complementarity, within a given area, between parvalbumin and calbindin expression across layers. To investigate the possible differences in the energetic metabolism of the cortical auditory areas, several metabolic markers were used: cytochrome oxidase and LDH1 were used as oxidative metabolism markers and LDH5 was used as glycolytic metabolism marker. The results obtained show a difference in the expression of enzymes involved in oxidative metabolism between areas. In the primary auditory area the oxidative metabolism markers were maximally expressed in layer IV. In contrast, higher order areas showed maximal staining in supragranular layers. The expression of LDH5 varied in patches, but did not differ between the different hierarchical auditory areas. The distribution of the two LDH enzymes isoforms also provides information about cellular aspects of metabolic organization, since neurons expressed the LDH1 isoform whereas astrocytes express primarily LDH5, but some astrocytes also contained the LDH1 isoform. This cellular distribution pattern supports the hypothesis of the existence of an astrocyte-neuron lactate shuttle, previously suggested in rodent studies, and in particular of lactate transfer from astrocytes, which produce lactate from the glucose obtained from the circulation, to neurons that use lactate as energy substrate. In conclusion, the hypothesis of parallel and hierarchical organization of the auditory areas can be supported by CaBPs, cytochrome oxidase and LDH1 distribution. Moreover, the two LDHs cellular distribution pattern support the hypothesis of an astrocyte-neuron lactate shuttle in human cortex.

Procalcitonin for reduced antibiotic exposure in ventilator-associated pneumonia: a randomised study.

In patients with ventilator-associated pneumonia (VAP), guidelines recommend antibiotic therapy adjustment according to microbiology results after 72 h. Circulating procalcitonin levels may provide evidence that facilitates the reduction of antibiotic therapy. In a multicentre, randomised, controlled trial, 101 patients with VAP were assigned to an antibiotic discontinuation strategy according to guidelines (control group) or to serum procalcitonin concentrations (procalcitonin group) with an antibiotic regimen selected by the treating physician. The primary end-point was antibiotic-free days alive assessed 28 days after VAP onset and analysed on an intent-to-treat basis. Procalcitonin determination significantly increased the number of antibiotic free-days alive 28 days after VAP onset (13 (2-21) days versus 9.5 (1.5-17) days). This translated into a reduction in the overall duration of antibiotic therapy of 27% in the procalcitonin group (p = 0.038). After adjustment for age, microbiology and centre effect, the rate of antibiotic discontinuation on day 28 remained higher in the procalcitonin group compared with patients treated according to guidelines (hazard rate 1.6, 95% CI 1.02-2.71). The number of mechanical ventilation-free days alive, intensive care unit-free days alive, length of hospital stay and mortality rate on day 28 for the two groups were similar. Serum procalcitonin reduces antibiotic therapy exposure in patients with ventilator associated pneumonia.

A tumor-specific cellular environment at the brain invasion border of adamantinomatous craniopharyngiomas.

Craniopharyngiomas (CP) are benign epithelial tumors of the sellar region and can be clinicopathologically distinguished into adamantinomatous (adaCP) and papillary (papCP) variants. Both subtypes are classified according to the World Health Organization grade I, but their irregular digitate brain infiltration makes any complete surgical resection difficult to obtain. Herein, we characterized the cellular interface between the tumor and the surrounding brain tissue in 48 CP (41 adaCP and seven papCP) compared to non-neuroepithelial tumors, i.e., 12 cavernous hemangiomas, 10 meningiomas, and 14 metastases using antibodies directed against glial fibrillary acid protein (GFAP), vimentin, nestin, microtubule-associated protein 2 (MAP2) splice variants, and tenascin-C. We identified a specific cell population characterized by the coexpression of nestin, MAP2, and GFAP within the invasion niche of the adamantinomatous subtype. This was especially prominent along the finger-like protrusions. A similar population of presumably astroglial precursors was not visible in other lesions under study, which characterize them as distinct histopathological feature of adaCP. Furthermore, the outer tumor cell layer of adaCP showed a distinct expression of MAP2, a novel finding helpful in the differential diagnosis of epithelial tumors in the sellar region. Our data support the hypothesis that adaCP, unlike other non-neuroepithelial tumors of the central nervous system, create a tumor-specific cellular environment at the tumor-brain junction. Whether this facilitates the characteristic infiltrative growth pattern or is the consequence of an activated Wnt signaling pathway, detectable in 90% of these tumors, will need further consideration.

Neuronal responses in mouse barrel cortex:Comparison of two signal discriminators

Barrels are discrete cytoarchitectonic neurons cluster located in the layer IV of the somatosensory¦cortex in mice brain. Each barrel is related to a specific whisker located on the mouse snout. The¦whisker-to-barrel pathway is a part of the somatosensory system that is intensively used to explore¦sensory activation induced plasticity in the cerebral cortex.¦Different recording methods exist to explore the cortical response induced by whisker deflection in¦the cortex of anesthetized mice. In this work, we used a method called the Single-Unit Analysis by¦which we recorded the extracellular electric signals of a single barrel neuron using a microelectrode.¦After recording the signal was processed by discriminators to isolate specific neuronal shape (action¦potentials).¦The objective of this thesis was to familiarize with the barrel cortex recording during whisker¦deflection and its theoretical background and to compare two different ways of discriminating and¦sorting cortical signal, the Waveform Window Discriminator (WWD) or the Spike Shape Discriminator (SSD).¦WWD is an electric module allowing the selection of specific electric signal shape. A trigger and a¦window potential level are set manually. During measurements, every time the electric signal passes¦through the two levels a dot is generated on time line. It was the method used in previous¦extracellular recording study in the Département de Biologie Cellulaire et de Morphologie (DBCM) in¦Lausanne.¦SSD is a function provided by the signal analysis software Spike2 (Cambridge Electronic Design). The¦neuronal signal is discriminated by a complex algorithm allowing the creation of specific templates.¦Each of these templates is supposed to correspond to a cell response profile. The templates are saved¦as a number of points (62 in this study) and are set for each new cortical location. During¦measurements, every time the cortical recorded signal corresponds to a defined number of templates¦points (60% in this study) a dot is generated on time line. The advantage of the SSD is that multiple¦templates can be used during a single stimulation, allowing a simultaneous recording of multiple¦signals.¦It exists different ways to represent data after discrimination and sorting. The most commonly used¦in the Single-Unit Analysis of the barrel cortex are the representation of the time between stimulation¦and the first cell response (the latency), the representation of the Response Magnitude (RM) after¦whisker deflection corrected for spontaneous activity and the representation of the time distribution¦of neuronal spikes on time axis after whisker stimulation (Peri-Stimulus Time Histogram, PSTH).¦The results show that the RMs and the latencies in layer IV were significantly different between the¦WWD and the SSD discriminated signal. The temporal distribution of the latencies shows that the¦different values were included between 6 and 60ms with no peak value for SSD while the WWD¦data were all gathered around a peak of 11ms (corresponding to previous studies). The scattered¦distribution of the latencies recorded with the SSD did not correspond to a cell response.¦The SSD appears to be a powerful tool for signal sorting but we do not succeed to use it for the¦Single-Unit Analysis extracellular recordings. Further recordings with different SSD templates settings¦and larger sample size may help to show the utility of this tool in Single-Unit Analysis studies.

Sweet sixteen for ANLS.

Since its introduction 16 years ago, the astrocyte-neuron lactate shuttle (ANLS) model has profoundly modified our understanding of neuroenergetics by bringing a cellular and molecular resolution. Praised or disputed, the concept has never ceased to attract attention, leading to critical advances and unexpected insights. Here, we summarize recent experimental evidence further supporting the main tenets of the model. Thus, evidence for distinct metabolic phenotypes between neurons (mainly oxidative) and astrocytes (mainly glycolytic) have been provided by genomics and classical metabolic approaches. Moreover, it has become clear that astrocytes act as a syncytium to distribute energy substrates such as lactate to active neurones. Glycogen, the main energy reserve located in astrocytes, is used as a lactate source to sustain glutamatergic neurotransmission and synaptic plasticity. Lactate is also emerging as a neuroprotective agent as well as a key signal to regulate blood flow. Characterization of monocarboxylate transporter regulation indicates a possible involvement in synaptic plasticity and memory. Finally, several modeling studies captured the implications of such findings for many brain functions. The ANLS model now represents a useful, experimentally based framework to better understand the coupling between neuronal activity and energetics as it relates to neuronal plasticity, neurodegeneration, and functional brain imaging.