Involvement of gap junctional intercellular communication in the pathophysiology of inflammation

SUMMARY Inflammation has evolved as a mechanism to defend the body against invading microorganisms and to respond to injury. It requires the coordinated response of a large number of cell types from the whole organism in a time- and space-dependent fashion. This coordination involves several cell-cell communication mechanisms. Exchange of humoral mediators such as cytokines is a major one. Moreover, direct contact between cells happens and plays a primordial role, for example when macrophages present antigens to lymphocytes. Contact between endothelial cells and leucocytes occurs when the latter cross the blood vessel barrier and transmigrate to the inflammatory site. A particular way by which cells communicate with each other in the course of inflammation, which at this time starts to gain attention, is the intercellular communication mediated by gap junctions. Gap junctions are channels providing a direct pathway (i.e. without transit through the extracellular space) for the diffusion of small molecules between adjacent cells. This process is known as gap junctional intercellular communication (GJIC). The general aim of this thesis was to study a possible involvement of GJIC in the pathophysiology of inflammation. A first part of the work was dedicated to study the implication of GJIC in the modification of vascular endothelial function by inflammation. In a second part, we were interested in the possible role of GJIC in the transmigration of neutrophil polymorphonuclear leucocytes through the endothelium. The main positive finding of this work is that acute inflammation preferentially modulates the expression of connexin 40 (Cx40), a gap junction protein specifically expressed in vascular endothelium. The modulation could be towards overexpression (aortic endothelium of septic rats) or towards downregulation (acutely inflamed mouse lung). We put a lot of efforts in search of possible functions of these modulations, in two directions: a potential protective role of Cx40 increased expression against sepsis-induced endothelial dysfunction, and a facilitating role of Cx40 decreased expression in neutrophil transmigration. To pursue both directions, it seemed logical to study the impact of Cx40 deletion using knock-out mice. Concerning the potential protective role of Cx40 overexpression we encountered a roadblock as we observed, in the aorta, a Cx40 downregulation in wild type mouse whereas Cx40 was upregulated in the rat. Regarding the second direction and using an in vivo approach, we observed that pulmonary neutrophil transmigration was not affected by the genetic deletion of Cx40. In spite of their negative nature, these results are the very first ones regarding the potential implication of GJIC concerning leucocyte transmigration in vivo. Because this process involves such tight cell-cell physical contacts, the hypothesis for a role of GJIC remains attractive.

Getting down to the core of homologous recombination.

In a paper in this week's issue of Science, Voloshin et al. (p. 868) show that a 20-amino acid peptide from RecA, a bacterial protein that repairs and recombines DNA, can mediate DNA strand exchange--one of the functions of the RecA protein. Stasiak discusses why this result is surprising and what the rest of the RecA protein is for.

Exome sequencing identifies INPPL1 mutations as a cause of opsismodysplasia.

Opsismodysplasia (OPS) is a severe autosomal-recessive chondrodysplasia characterized by pre- and postnatal micromelia with extremely short hands and feet. The main radiological features are severe platyspondyly, squared metacarpals, delayed skeletal ossification, and metaphyseal cupping. In order to identify mutations causing OPS, a total of 16 cases (7 terminated pregnancies and 9 postnatal cases) from 10 unrelated families were included in this study. We performed exome sequencing in three cases from three unrelated families and only one gene was found to harbor mutations in all three cases: inositol polyphosphate phosphatase-like 1 (INPPL1). Screening INPPL1 in the remaining cases identified a total of 12 distinct INPPL1 mutations in the 10 families, present at the homozygote state in 7 consanguinous families and at the compound heterozygote state in the 3 remaining families. Most mutations (6/12) resulted in premature stop codons, 2/12 were splice site, and 4/12 were missense mutations located in the catalytic domain, 5-phosphatase. INPPL1 belongs to the inositol-1,4,5-trisphosphate 5-phosphatase family, a family of signal-modulating enzymes that govern a plethora of cellular functions by regulating the levels of specific phosphoinositides. Our finding of INPPL1 mutations in OPS, a severe spondylodysplastic dysplasia with major growth plate disorganization, supports a key and specific role of this enzyme in endochondral ossification.

Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors.

Tumor progression is facilitated by regulatory T cells (Treg) and restricted by effector T cells. In this study, we document parallel regulation of CD8(+) T cells and Foxp3(+) Tregs by programmed death-1 (PD-1, PDCD1). In addition, we identify an additional role of CTL antigen-4 (CTLA-4) inhibitory receptor in further promoting dysfunction of CD8(+) T effector cells in tumor models (CT26 colon carcinoma and ID8-VEGF ovarian carcinoma). Two thirds of CD8(+) tumor-infiltrating lymphocytes (TIL) expressed PD-1, whereas one third to half of CD8(+) TIL coexpressed PD-1 and CTLA-4. Double-positive (PD-1(+)CTLA-4(+)) CD8(+) TIL had characteristics of more severe dysfunction than single-positive (PD-1(+) or CTLA-4(+)) TIL, including an inability to proliferate and secrete effector cytokines. Blockade of both PD-1 and CTLA-4 resulted in reversal of CD8(+) TIL dysfunction and led to tumor rejection in two thirds of mice. Double blockade was associated with increased proliferation of antigen-specific effector CD8(+) and CD4(+) T cells, antigen-specific cytokine release, inhibition of suppressive functions of Tregs, and upregulation of key signaling molecules critical for T-cell function. When used in combination with GVAX vaccination (consisting of granulocyte macrophage colony-stimulating factor-expressing irradiated tumor cells), inhibitory pathway blockade induced rejection of CT26 tumors in 100% of mice and ID8-VEGF tumors in 75% of mice. Our study indicates that PD-1 signaling in tumors is required for both suppressing effector T cells and maintaining tumor Tregs, and that PD-1/PD-L1 pathway (CD274) blockade augments tumor inhibition by increasing effector T-cell activity, thereby attenuating Treg suppression. Cancer Res; 73(12); 3591-603. ©2013 AACR.

Effect of Vagus Nerve Stimulation in an Adult Patient with Dravet Syndrome: Contribution to Sudden Unexpected Death in Epilepsy Risk Reduction?.

We report on a patient who developed, from 5 months of age, multiple seizure types, including myoclonic, associated with severe psychomotor delay, leading to the diagnosis of Dravet syndrome. Over the years, he developed refractory epilepsy and was implanted with a vagus nerve stimulator at the age of 19. After 3 months, he experienced a progressive improvement of partial and generalized seizures, with a >90% reduction, and better alertness. This meaningful clinical improvement is discussed in the light of the sudden unexpected death in epilepsy risk, which is high in this setting, and seems remarkably diminished in our patient in view of the reduction of generalized convulsions.

The Role of Interleukin-2 in Memory CD8 Cell Differentiation

The current literature on the role of interleukin (IL)-2 in memory CD8(+) T-cell differentiation indicates a significant contribution of IL-2 during primary and also secondary expansion of CD8(+) T cells. IL-2 seems to be responsible for optimal expansion and generation of effector functions following primary antigenic challenge. As the magnitude of T-cell expansion determines the numbers of memory CD8(+) T cells surviving after pathogen elimination, these events influence memory cell generation. Moreover, during the contraction phase of an immune response where most antigen-specific CD8(+) T cells disappear by apoptosis, IL-2 signals are able to rescue CD8(+) T cells from cell death and provide a durable increase in memory CD8(+) T-cell counts. At the memory stage, CD8(+) T-cell frequencies can be boosted by administration of exogenous IL-2. Significantly, only CD8(+) T cells that have received IL-2 signals during initial priming are able to mediate efficient secondary expansion following renewed antigenic challenge. Thus, IL-2 signals during different phases of an immune response are key in optimizing CD8(+) T-cell functions, thereby affecting both primary and secondary responses of these T cells.

Characterization of surface functional groups present on laboratory-generated and ambient aerosol particles by means of heterogeneous titration reactions

A Knudsen flow reactor has been used to quantify surface functional groups on aerosols collected in the field. This technique is based on a heterogeneous titration reaction between a probe gas and a specific functional group on the particle surface. In the first part of this work, the reactivity of different probe gases on laboratory-generated aerosols (limonene SOA, Pb(NO3)2, Cd(NO3)2) and diesel reference soot (SRM 2975) has been studied. Five probe gases have been selected for the quantitative determination of important functional groups: N(CH3)3 (for the titration of acidic sites), NH2OH (for carbonyl functions), CF3COOH and HCl (for basic sites of different strength), and O3 (for oxidizable groups). The second part describes a field campaign that has been undertaken in several bus depots in Switzerland, where ambient fine and ultrafine particles were collected on suitable filters and quantitatively investigated using the Knudsen flow reactor. Results point to important differences in the surface reactivity of ambient particles, depending on the sampling site and season. The particle surface appears to be multi-functional, with the simultaneous presence of antagonistic functional groups which do not undergo internal chemical reactions, such as acid-base neutralization. Results also indicate that the surface of ambient particles was characterized by a high density of carbonyl functions (reactivity towards NH2OH probe in the range 0.26-6 formal molecular monolayers) and a low density of acidic sites (reactivity towards N(CH3)3 probe in the range 0.01-0.20 formal molecular monolayer). Kinetic parameters point to fast redox reactions (uptake coefficient ?0>10-3 for O3 probe) and slow acid-base reactions (?0<10-4 for N(CH3)3 probe) on the particle surface. [Authors]

The puzzles of the prokineticin 2 pathway in human reproduction.

Prokineticin, 1 (PROK1) and prokineticin 2 (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. MIT-1 was initially identified as a non-toxic constituent in the venom of the black mamba snake (Dendroaspis polylepis) (Joubert and Strydom, 1980) while Bv8 was identified in the skin secretion of the toad, Bombina variegate (Mollay et al., 1999). All three homologs stimulate gastrointestinal motility thus accounting for their family name "prokineticins" (Schweitz et al., 1990, 1999). However, since its initial description, both PROK1 and PROK2 have been found to regulate a dazzling array of biological functions throughout the body. In particular, PROK1 acts as a potent angiogenic mitogen on endocrine vascular epithelium, thus earning its other name, Endocrine gland-vascular endothelial factor (EG-VEGF) (LeCouter et al., 2002). In contrast, the PROK2 signaling pathway is a critical regulator of olfactory bulb morphogenesis and sexual maturation in mammals and this function is the focus of this review.

Origins, evolution, and phenotypic impact of new genes.

Ever since the pre-molecular era, the birth of new genes with novel functions has been considered to be a major contributor to adaptive evolutionary innovation. Here, I review the origin and evolution of new genes and their functions in eukaryotes, an area of research that has made rapid progress in the past decade thanks to the genomics revolution. Indeed, recent work has provided initial whole-genome views of the different types of new genes for a large number of different organisms. The array of mechanisms underlying the origin of new genes is compelling, extending way beyond the traditionally well-studied source of gene duplication. Thus, it was shown that novel genes also regularly arose from messenger RNAs of ancestral genes, protein-coding genes metamorphosed into new RNA genes, genomic parasites were co-opted as new genes, and that both protein and RNA genes were composed from scratch (i.e., from previously nonfunctional sequences). These mechanisms then also contributed to the formation of numerous novel chimeric gene structures. Detailed functional investigations uncovered different evolutionary pathways that led to the emergence of novel functions from these newly minted sequences and, with respect to animals, attributed a potentially important role to one specific tissue--the testis--in the process of gene birth. Remarkably, these studies also demonstrated that novel genes of the various types significantly impacted the evolution of cellular, physiological, morphological, behavioral, and reproductive phenotypic traits. Consequently, it is now firmly established that new genes have indeed been major contributors to the origin of adaptive evolutionary novelties.

Brain Connectivity Analysis in Children. Effects of Prematurity. and Prenatal Growth Restriction

Introduction: Survival of children born prematurely or with very low birth weight has increased dramatically, but the long term developmental outcome remains unknown. Many children have deficits in cognitive capacities, in particular involving executive domains and those disabilities are likely to involve a central nervous system deficit. To understand their neurostructural origin, we use DTI. Structurally segregated and functionally regions of the cerebral cortex are interconnected by a dense network of axonal pathways. We noninvasively map these pathways across cortical hemispheres and construct normalized structural connection matrices derived from DTI MR tractography. Group comparisons of brain connectivity reveal significant changes in fiber density in case of children with poor intrauterine grown and extremely premature children (gestational age<28 weeks at birth) compared to control subjects. This changes suggest a link between cortico-axonal pathways and the central nervous system deficit. Methods: Sixty premature born infants (5-6 years old) were scanned on clinical 3T scanner (Magnetom Trio, Siemens Medical Solutions, Erlangen, Germany) at two hospitals (HUG, Geneva and CHUV, Lausanne). For each subject, T1-weighted MPRAGE images (TR/TE=2500/2.91,TI=1100, resolution=1x1x1mm, matrix=256x154) and DTI images (30 directions, TR/TE=10200/107, in-plane resolution=1.8x1.8x2mm, 64 axial, matrix=112x112) were acquired. Parent(s) provided written consent on prior ethical board approval. The extraction of the Whole Brain Structural Connectivity Matrix was performed following (Cammoun, 2009 and Hagmann, 2008). The MPARGE images were registered using an affine registration to the non-weighted-DTI and WM-GM segmentation performed on it. In order to have equal anatomical localization among subjects, 66 cortical regions with anatomical landmarks were created using the curvature information, i.e. sulcus and gyrus (Cammoun et al, 2007; Fischl et al, 2004; Desikan et al, 2006) with freesurfer software (http://surfer.nmr.mgh.harvard.edu/). Tractography was performed in WM using an algorithm especially designed for DTI/DSI data (Hagmann et al., 2007) and both information were then combined in a matrix. Each row and column of the matrix corresponds to a particular ROI. Each cell of index (i,j) represents the fiber density of the bundle connecting the ROIs i and j. Subdividing each cortical region, we obtained 4 Connectivity Matrices of different resolution (33, 66, 125 and 250 ROI/hemisphere) for each subject . Subjects were sorted in 3 different groups, namely (1) control, (2) Intrauterine Growth Restriction (IUGR), (3) Extreme Prematurity (EP), depending on their gestational age, weight and percentile-weight score at birth. Group-to-group comparisons were performed between groups (1)-(2) and (1)-(3). The mean age at examination of the three groups were similar. Results: Quantitative analysis were performed between groups to determine fibers density differences. For each group, a mean connectivity matrix with 33ROI/hemisphere resolution was computed. On the other hand, for all matrix resolutions (33,66,125,250 ROI/hemisphere), the number of bundles were computed and averaged. As seen in figure 1, EP and IUGR subjects present an overall reduction of fibers density in both interhemispherical and intrahemispherical connections. This is given quantitatively in table 1. IUGR subjects presents a higher percentage of missing fiber bundles than EP when compared to control subjects (~16% against 11%). When comparing both groups to control subjects, for the EP subjects, the occipito-parietal regions seem less interhemispherically connected whilst the intrahemispherical networks present lack of fiber density in the lymbic system. Children born with IUGR, have similar reductions in interhemispherical connections than the EP. However, the cuneus and precuneus connections with the precentral and paracentral lobe are even lower than in the case of the EP. For the intrahemispherical connections the IUGR group preset a loss of fiber density between the deep gray matter structures (striatum) and the frontal and middlefrontal poles, connections typically involved in the control of executive functions. For the qualitative analysis, a t-test comparing number of bundles (p-value<0.05) gave some preliminary significant results (figure 2). Again, even if both IUGR and EP appear to have significantly less connections comparing to the control subjects, the IUGR cohort seems to present a higher lack of fiber density specially relying the cuneus, precuneus and parietal areas. In terms of fiber density, preliminary Wilcoxon tests seem to validate the hypothesis set by the previous analysis. Conclusions: The goal of this study was to determine the effect of extreme prematurity and poor intrauterine growth on neurostructural development at the age of 6 years-old. This data indicates that differences in connectivity may well be the basis for the neurostructural and neuropsychological deficit described in these populations in the absence of overt brain lesions (Inder TE, 2005; Borradori-Tolsa, 2004; Dubois, 2008). Indeed, we suggest that IUGR and prematurity leads to alteration of connectivity between brain structures, especially in occipito-parietal and frontal lobes for EP and frontal and middletemporal poles for IUGR. Overall, IUGR children have a higher loss of connectivity in the overall connectivity matrix than EP children. In both cases, the localized alteration of connectivity suggests a direct link between cortico-axonal pathways and the central nervous system deficit. Our next step is to link these connectivity alterations to the performance in executive function tests.

Atomic diversity, molecular diversity, and chemical diversity: the concept of chemodiversity.

This minireview is meant as an introduction to the following paper. To this end, it presents the general background against which the joint paper should be understood. The first objective of the present paper is thus to clarify some concepts and related terminology, drawing a clear distinction between i) atomic diversity (i.e., atomic-property space), ii) molecular or macromolecular diversity (i.e., molecular- or macromolecular-property spaces), and iii) chemical diversity (i.e., chemical-diversity space). The first refers to the various electronic states an atom can occupy. The second encompasses the conformational and property spaces of a given (macro)molecule. The third pertains to the diversity in structure and properties exhibited by a library or a supramolecular assembly of different chemical compounds. The ground is thus laid for the content of the joint paper, which pertains to case ii, to be placed in its broader chemodiversity context. The second objective of this paper is to point to the concepts of chemodiversity and biodiversity as forming a continuum. Chemodiversity is indeed the material substratum of organisms. In other words, chemodiversity is the material condition for life to emerge and exist. Increasing our knowledge of chemodiversity is thus a condition for a better understanding of life as a process.

Novel action of a wake-promoting neuropeptide in hippocampus : inhibition of nmda receptor function at excitatory synapses through orexin-a

One of the most intriguing functions of the brain is the ability to learn and memorize. The mechanism through which memory and learning are expressed requires the activation of NMDA receptors (NMDARs). These molecular entities are placed at the postsynaptic density of excitatory synapses and their function is tightly controlled by the actions of several modulators at the extracellular, intracellular and pore sites. A large part of the intracellular modulation comes from the action of G-protein coupled receptors (GPCRs). Through intracellular cascades typically involving kinases and phosphatases, GPCRs potentiate or inhibit NMDARs, controlling the conductive state but also the trafficking within the synapse. The GPCRs are involved in the modulation of a variety of brain functions. Many of them control cognition, memory and learning performance, therefore, their effects on NMDARs are extensively studied. The orexinergic system signals through GPCRs and it is well known for the regulation of waking, feeding, reward and autonomic functions. Moreover, it is involved in potentiating hippocampus-related cognitive tasks. Orexin receptors and fibers are present within the hippocampus, but whether these directly modulate hippocampal cells and synapses has not yet been determined. During my thesis, I studied orexinergic actions on excitatory synaptic transmission via whole-cell patch-clamp recordings in rat acute hippocampal slices. I observed that exogenously applied orexin-A (ox-A) exerted a strong inhibitory action on NMDAR-mediated synaptic potentials at mossy fiber (MF)-CA3 synapses, by postsynaptically activating orexin-2 receptors, a minor inhibition at Schaffer collateral-CAl synapses and did not affect other synapses with the CA3 area. Moreover, I demonstrated that the susceptibility of NMDARs to ox- A depends on the tone of endogenous orexin known to fluctuate during the day-night cycle. In fact, in slices prepared during the active period of the rats, when endogenous orexin levels are high, NMDAR-currents were not affected by exogenously applied ox-A. The inhibitory effect of ox-A was, however, reverted when interfering with the orexinergic system through intraperitoneal injections of almorexant, a dual orexin receptor antagonist, during the active phase prior to slice preparation. This thesis work suggests that the orexinergic system regulates NMDAR-dependent information flow through select hippocampal pathways depending on the time-of-day. The specific orexinergic modulation of NMDARs at MFs dampens the excitability of the hippocampal circuit and could impede the mechanisms related to memory formation, possibly also following extended periods of waking. -- La capacité d'apprentissage et de mémorisation est une des fonctions les plus intrigantes de notre cerveau. Il a été montré qu'elles requièrent l'activation des récepteurs NMDA (NMDARs). Ces entités moléculaires sont présentes au niveau de la densité post-synaptique des synapses excitatrices et leur fonction est étroitement contrôlée par l'action de nombreux modulateurs au niveau extracellulaire, intracellulaire et membranaire de ces récepteurs. Une grande partie de la modulation intracellulaire s'effectue via l'action de récepteurs couplés aux protéines G (GPCRs). Grace à leurs cascades intracellulaires typiquement impliquant des kinases et des phosphatases, les GPCRs favorisent l'activation ou l'inhibition des NMDARs, contrôlant ainsi leur perméabilité mais aussi leur mouvement à la synapse. Les GPCRs sont impliquées dans de nombreuses fonctions cérébrales telles que la cognition, la mémoire ainsi que la capacité d'apprentissage c'est pour cela que leurs effets sur les NMDARs sont très étudiés. Le système orexinergique fait intervenir ces GPCRs et est connu par son rôle dans la régulation de fonctions physiologiques telles que l'éveil, la prise alimentaire, la récompense ainsi que d'autres fonctions du système nerveux autonome. De plus, ce système est impliqué dans la régulation de tâches cognitives liées à l'hippocampe. Bien que les fibres et les récepteurs à l'orexine soient présents dans l'hippocampe, leur mécanisme d'action sur les cellules et les synapses de l'hippocampe n'a pas encore été élucidé. Durant ma thèse, je me suis intéressée aux effets de l'orexine sur la transmission synaptique excitatrice en utilisant la méthode d'enregistrement en patch-clamp en configuration cellule entière sur des tranches aiguës d'hippocampes de rats. J'ai observé que l'application exogène d'orexine A d'une part inhibe fortement les courants synaptiques dépendants de l'activation des NMDARs au niveau de la synapse entre les fibres moussues et CA3 via l'activation post-synaptique des orexine récepteurs 2 mais d'autre part n'inhibe que de façon mineure la synapse entre les collatérales de Schaffer et CAI et n'affecte pas les autres synapses impliquant CA3. J'ai également démontré que la sensibilité des NMDARs à l'orexine A dépend de sa concentration endogène qui fluctue durant le cycle éveil-sommeil. En effet, lorsque les coupes d'hippocampes sont préparées durant la période active de l'animal correspondant à un niveau endogène d'orexine élevé, l'application exogène d'orexine A n'a aucun effet sur les courants dépendants de l'activation des NMDARs. Cependant, l'injection dans le péritoine, durant la phase active de l'animal, d'un antagoniste des orexine récepteurs, l'almorexant, va supprimer l'effet inhibiteur de l'orexine A. Les résultats de ma thèse suggèrent donc que le système orexinergique module les informations véhiculées par les NMDARs via des voies de signalisation sélectives de l'hippocampe en fonction du moment de la journée. La modulation orexinergique des NMDARs au niveau des fibres moussues diminue ainsi l'excitabilité du circuit hippocampal et pourrait entraver les mécanismes liés à la formation de la mémoire, potentiellement après de longues périodes d'éveil.

Molecular bases of circadian rhythmicity in renal physiology and pathology.

The physiological processes that maintain body homeostasis oscillate during the day. Diurnal changes characterize kidney functions, comprising regulation of hydro-electrolytic and acid-base balance, reabsorption of small solutes and hormone production. Renal physiology is characterized by 24-h periodicity and contributes to circadian variability of blood pressure levels, related as well to nychthemeral changes of sodium sensitivity, physical activity, vascular tone, autonomic function and neurotransmitter release from sympathetic innervations. The circadian rhythmicity of body physiology is driven by central and peripheral biological clockworks and entrained by the geophysical light/dark cycle. Chronodisruption, defined as the mismatch between environmental-social cues and physiological-behavioral patterns, causes internal desynchronization of periodic functions, leading to pathophysiological mechanisms underlying degenerative, immune related, metabolic and neoplastic diseases. In this review we will address the genetic, molecular and anatomical elements that hardwire circadian rhythmicity in renal physiology and subtend disarray of time-dependent changes in renal pathology.

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.