Mechanisms underlying functional recovery after in vivo focal cerebral ischemia : from preconditioning to diffusion MRI

Version abregée L'ischémie cérébrale est la troisième cause de mort dans les pays développés, et la maladie responsable des plus sérieux handicaps neurologiques. La compréhension des bases moléculaires et anatomiques de la récupération fonctionnelle après l'ischémie cérébrale est donc extrêmement importante et représente un domaine d'intérêt crucial pour la recherche fondamentale et clinique. Durant les deux dernières décennies, les chercheurs ont tenté de combattre les effets nocifs de l'ischémie cérébrale à l'aide de substances exogènes qui, bien que testées avec succès dans le domaine expérimental, ont montré un effet contradictoire dans l'application clinique. Une approche différente mais complémentaire est de stimuler des mécanismes intrinsèques de neuroprotection en utilisant le «modèle de préconditionnement» : une brève insulte protège contre des épisodes d'ischémie plus sévères à travers la stimulation de voies de signalisation endogènes qui augmentent la résistance à l'ischémie. Cette approche peut offrir des éléments importants pour clarifier les mécanismes endogènes de neuroprotection et fournir de nouvelles stratégies pour rendre les neurones et la glie plus résistants à l'attaque ischémique cérébrale. Dans un premier temps, nous avons donc étudié les mécanismes de neuroprotection intrinsèques stimulés par la thrombine, un neuroprotecteur «préconditionnant» dont on a montré, à l'aide de modèles expérimentaux in vitro et in vivo, qu'il réduit la mort neuronale. En appliquant une technique de microchirurgie pour induire une ischémie cérébrale transitoire chez la souris, nous avons montré que la thrombine peut stimuler les voies de signalisation intracellulaire médiées par MAPK et JNK par une approche moléculaire et l'analyse in vivo d'un inhibiteur spécifique de JNK (L JNK) .Nous avons également étudié l'impact de la thrombine sur la récupération fonctionnelle après une attaque et avons pu démontrer que ces mécanismes moléculaires peuvent améliorer la récupération motrice. La deuxième partie de cette étude des mécanismes de récupération après ischémie cérébrale est basée sur l'investigation des bases anatomiques de la plasticité des connections cérébrales, soit dans le modèle animal d'ischémie transitoire, soit chez l'homme. Selon des résultats précédemment publiés par divers groupes ,nous savons que des mécanismes de plasticité aboutissant à des degrés divers de récupération fonctionnelle sont mis enjeu après une lésion ischémique. Le résultat de cette réorganisation est une nouvelle architecture fonctionnelle et structurelle, qui varie individuellement selon l'anatomie de la lésion, l'âge du sujet et la chronicité de la lésion. Le succès de toute intervention thérapeutique dépendra donc de son interaction avec la nouvelle architecture anatomique. Pour cette raison, nous avons appliqué deux techniques de diffusion en résonance magnétique qui permettent de détecter les changements de microstructure cérébrale et de connexions anatomiques suite à une attaque : IRM par tenseur de diffusion (DT-IR1V) et IRM par spectre de diffusion (DSIRM). Grâce à la DT-IRM hautement sophistiquée, nous avons pu effectuer une étude de follow-up à long terme chez des souris ayant subi une ischémie cérébrale transitoire, qui a mis en évidence que les changements microstructurels dans l'infarctus ainsi que la modification des voies anatomiques sont corrélés à la récupération fonctionnelle. De plus, nous avons observé une réorganisation axonale dans des aires où l'on détecte une augmentation d'expression d'une protéine de plasticité exprimée dans le cône de croissance des axones (GAP-43). En appliquant la même technique, nous avons également effectué deux études, rétrospective et prospective, qui ont montré comment des paramètres obtenus avec DT-IRM peuvent monitorer la rapidité de récupération et mettre en évidence un changement structurel dans les voies impliquées dans les manifestations cliniques. Dans la dernière partie de ce travail, nous avons décrit la manière dont la DS-IRM peut être appliquée dans le domaine expérimental et clinique pour étudier la plasticité cérébrale après ischémie. Abstract Ischemic stroke is the third leading cause of death in developed countries and the disease responsible for the most serious long-term neurological disability. Understanding molecular and anatomical basis of stroke recovery is, therefore, extremely important and represents a major field of interest for basic and clinical research. Over the past 2 decades, much attention has focused on counteracting noxious effect of the ischemic insult with exogenous substances (oxygen radical scavengers, AMPA and NMDA receptor antagonists, MMP inhibitors etc) which were successfully tested in the experimental field -but which turned out to have controversial effects in clinical trials. A different but complementary approach to address ischemia pathophysiology and treatment options is to stimulate and investigate intrinsic mechanisms of neuroprotection using the "preconditioning effect": applying a brief insult protects against subsequent prolonged and detrimental ischemic episodes, by up-regulating powerful endogenous pathways that increase resistance to injury. We believe that this approach might offer an important insight into the molecular mechanisms responsible for endogenous neuroprotection. In addition, results from preconditioning model experiment may provide new strategies for making brain cells "naturally" more resistant to ischemic injury and accelerate their rate of functional recovery. In the first part of this work, we investigated down-stream mechanisms of neuroprotection induced by thrombin, a well known neuroprotectant which has been demonstrated to reduce stroke-induced cell death in vitro and in vivo experimental models. Using microsurgery to induce transient brain ischemia in mice, we showed that thrombin can stimulate both MAPK and JNK intracellular pathways through a molecular biology approach and an in vivo analysis of a specific kinase inhibitor (L JNK1). We also studied thrombin's impact on functional recovery demonstrating that these molecular mechanisms could enhance post-stroke motor outcome. The second part of this study is based on investigating the anatomical basis underlying connectivity remodeling, leading to functional improvement after stroke. To do this, we used both a mouse model of experimental ischemia and human subjects with stroke. It is known from previous data published in literature, that the brain adapts to damage in a way that attempts to preserve motor function. The result of this reorganization is a new functional and structural architecture, which will vary from patient to patient depending on the anatomy of the damage, the biological age of the patient and the chronicity of the lesion. The success of any given therapeutic intervention will depend on how well it interacts with this new architecture. For this reason, we applied diffusion magnetic resonance techniques able to detect micro-structural and connectivity changes following an ischemic lesion: diffusion tensor MRI (DT-MRI) and diffusion spectrum MRI (DS-MRI). Using DT-MRI, we performed along-term follow up study of stroke mice which showed how diffusion changes in the stroke region and fiber tract remodeling is correlating with stroke recovery. In addition, axonal reorganization is shown in areas of increased plasticity related protein expression (GAP 43, growth axonal cone related protein). Applying the same technique, we then performed a retrospective and a prospective study in humans demonstrating how specific DTI parameters could help to monitor the speed of recovery and show longitudinal changes in damaged tracts involved in clinical symptoms. Finally, in the last part of this study we showed how DS-MRI could be applied both to experimental and human stroke and which perspectives it can open to further investigate post stroke plasticity.

Effect of repetitive arm cycling following botulinum toxin injection for poststroke spasticity: evidence from FMRI.

BACKGROUND AND OBJECTIVE: Investigations were performed to establish if repetitive arm cycling training enhances the antispastic effect of intramuscular botulinum toxin (BTX) injections in postischemic spastic hemiparesis. Effects on cerebral activation were evaluated by functional magnetic resonance imaging (fMRI). METHODS: Eight chronic spastic hemisyndrome patients (49 ± 10 years) after middle cerebral artery infarction (5.5 ± 2.7 years) were investigated. BTX was injected into the affected arm twice, 6 months apart. Spasticity was assessed using the Ashworth Scale and range of motion before and 3 months after BTX injections. Images were analyzed using Brain Voyager QX 1.8, and fMRI signal changes were corrected for multiple comparisons. RESULTS: During passive movements of affected and nonaffected hands, fMRI activity was increased bilaterally in the sensorimotor cortex (MISI), secondary somatosensory areas (SII), and supplementary motor area predominantly in the contralesional hemisphere, compared with the rest. Following repetitive arm cycling, fMRI activity increased further in MISI of the lesioned hemisphere and SII of the contralesional hemisphere. For patients with residual motor activity, treatment-related fMRI activity increases were associated with reduced spasticity; in completely plegic patients, there was no fMRI activity change in SII but increased spasticity after training. CONCLUSION: Increased activity in SII of the contralesional hemisphere and in MISI of the lesioned hemisphere reflect a treatment-induced effect in the paretic arm. It is hypothesized that the increased BOLD activity results from increased afferent information related to the antispastic BTX effect reinforced by training.

Contribution of neural networks to Alzheimer disease's progression.

The neuropathology of Alzheimer disease is characterized by senile plaques, neurofibrillary tangles and cell death. These hallmarks develop according to the differential vulnerability of brain networks, senile plaques accumulating preferentially in the associative cortical areas and neurofibrillary tangles in the entorhinal cortex and the hippocampus. We suggest that the main aetiological hypotheses such as the beta-amyloid cascade hypothesis or its variant, the synaptic beta-amyloid hypothesis, will have to consider neural networks not just as targets of degenerative processes but also as contributors of the disease's progression and of its phenotype. Three domains of research are highlighted in this review. First, the cerebral reserve and the redundancy of the network's elements are related to brain vulnerability. Indeed, an enriched environment appears to increase the cerebral reserve as well as the threshold of disease's onset. Second, disease's progression and memory performance cannot be explained by synaptic or neuronal loss only, but also by the presence of compensatory mechanisms, such as synaptic scaling, at the microcircuit level. Third, some phenotypes of Alzheimer disease, such as hallucinations, appear to be related to progressive dysfunction of neural networks as a result, for instance, of a decreased signal to noise ratio, involving a diminished activity of the cholinergic system. Overall, converging results from studies of biological as well as artificial neural networks lead to the conclusion that changes in neural networks contribute strongly to Alzheimer disease's progression.

Changes of MAP2 phosphorylation during brain development.

The microtubule-associated protein MAP2 is essential for development of early neuronal morphology and maintenance of adult neuronal morphology. Several splice variants exist, MAP2a-d, with a lack of MAP2a in cat brain. MAP2 is widely used as a neuronal marker. In this study we compared five monoclonal antibodies (MAbs) against MAP2. They show differences in the immunocytochemical distribution of MAP2 isoforms during development of the visual cortex and cerebellum of the cat. Local and temporal differences were seen with MAb AP18, an antibody directed against a phosphorylation-dependent epitope near the N-terminal end. In large pyramidal dendrites in visual cortex, the AP18 epitope remained in parts immunoreactive after treatment with alkaline phosphatase. Three MAbs, AP14, MT-01, and MT-02, recognized the central region of the MAP2b molecule, which is not present in MAP2c and 2d, and reacted with phosphorylation-independent epitopes. During the first postnatal week the immunostaining in cerebellum differed between antibodies in that some cellular elements in external and internal granular layers and Purkinje cells were stained to various degrees, whereas at later stages staining patterns were similar. At early stages, antibody MT-02 stained cell bodies and dendrites in cerebral cortex and cerebellum. With progressing maturation, immunoreactivity became restricted to distal parts of apical dendrites of pyramidal cells and was absent from perikarya and finer proximal dendrites in cortex. MT-02 did not stain MAP2 in cerebellum of adult animals. This study demonstrates that the immunocytochemical detection of MAP2 depends on modifications such as phosphorylation and conformational changes of the molecule, and that MAP2 staining patterns differ between MAbs. Phosphorylation and specific conformations in the molecule may be essential for modulating function and molecular stability of MAP2, and monoclonal antibodies against such sites may provide tools for studying the functional role of modifications.

Population receptive field estimates of human auditory cortex.

Here we describe a method for measuring tonotopic maps and estimating bandwidth for voxels in human primary auditory cortex (PAC) using a modification of the population Receptive Field (pRF) model, developed for retinotopic mapping in visual cortex by Dumoulin and Wandell (2008). The pRF method reliably estimates tonotopic maps in the presence of acoustic scanner noise, and has two advantages over phase-encoding techniques. First, the stimulus design is flexible and need not be a frequency progression, thereby reducing biases due to habituation, expectation, and estimation artifacts, as well as reducing the effects of spatio-temporal BOLD nonlinearities. Second, the pRF method can provide estimates of bandwidth as a function of frequency. We find that bandwidth estimates are narrower for voxels within the PAC than in surrounding auditory responsive regions (non-PAC).

An Ion Transport-Independent Role for the Cation-Chloride Cotransporter KCC2 in Dendritic Spinogenesis In Vivo.

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport function.

Cerebral aneurysm exclusion by CT angiography based on subarachnoid hemorrhage pattern: a retrospective study

Contexte : Identifier les patients avec une hémorragie sous-arachnoïdienne spontanée chez qui l'angio-CT suffit pour exclure des anévrysmes rompus.Méthodes : Une étude rétrospective a été effectuée de tous les patients avec une hémorragie sous-arachnoïdienne qui ont eu un angio-CT ainsi qu'une angiographie par cathéter dans le but d'exclure un anévrysme. Les cas négatifs de l'angio-CT (sans anévrysmes) ont été classés d'après leur schéma hémorragique au CT dans les catégories suivantes : « anévrysmale », « périmésencéphalique » puis « sans hémorragie ».Résultats : Deux-cent-quarante-et-un patients ont été inclus. Une sensibilité de 96.4% et une spécificité de 96.0% ont été observée pour l'exclusion d'anévrysmes par l'angio-CT. Parmi les 78 cas négatifs de l'angio-CT, chacun des 35 cas avec un motif hémorragique périmésencéphalique ou sans hémorragie au CT n'ont pas eu d'anévrysmes démontrés à l'angiographie par cathéter.Conclusions: L'angio-CT est fiable pour exclure les anévrysmes rompus lorsqu'un motif hémorragique périmésencéphalique ou pas d'hémorragie sont visibles au CT à une semaine depuis le début des symptômes.

New pool of cortical interneuron precursors in the early postnatal dorsal white matter.

The migration of cortical γ-aminobutyric acidergic interneurons has been extensively studied in rodent embryos, whereas few studies have documented their postnatal migration. Combining in vivo analysis together with time-lapse imaging on cortical slices, we explored the origin and migration of cortical interneurons during the first weeks of postnatal life. Strikingly, we observed that a large pool of GAD65-GFP-positive cells accumulate in the dorsal white matter region during the first postnatal week. Part of these cells divides and expresses the transcription factor paired box 6 indicating the presence of local transient amplifying precursors. The vast majority of these cells are immature interneurons expressing the neuronal marker doublecortin and partly the calcium-binding protein calretinin. Time-lapse imaging reveals that GAD65-GFP-positive neurons migrate from the white matter pool into the overlying anterior cingulate cortex (aCC). Some interneurons in the postnatal aCC express the same immature neuronal markers suggesting ongoing migration of calretinin-positive interneurons. Finally, bromodeoxyuridine incorporation experiments confirm that a small fraction of interneurons located in the aCC are generated during the early postnatal period. These results altogether reveal that at postnatal ages, the dorsal white matter contains a pool of interneuron precursors that divide and migrate into the aCC.

Electromyographic, cerebral, and muscle hemodynamic responses during intermittent, isometric contractions of the biceps brachii at three submaximal intensities.

This study examined the electromyographic, cerebral and muscle hemodynamic responses during intermittent isometric contractions of biceps brachii at 20, 40, and 60% of maximal voluntary contraction (MVC). Eleven volunteers completed 2 min of intermittent isometric contractions (12/min) at an elbow angle of 90° interspersed with 3 min rest between intensities in systematic order. Surface electromyography (EMG) was recorded from the right biceps brachii and near infrared spectroscopy (NIRS) was used to simultaneously measure left prefrontal and right biceps brachii oxyhemoglobin (HbO2), deoxyhemoglobin (HHb), and total hemoglobin (Hbtot). Transcranial Doppler ultrasound was used to measure middle cerebral artery velocity (MCAv) bilaterally. Finger photoplethysmography was used to record beat-to-beat blood pressure and heart rate. EMG increased with force output from 20 to 60% MVC (P < 0.05). Cerebral HbO2 and Hbtot increased while HHb decreased during contractions with differences observed between 60% vs. 40% and 20% MVC (P < 0.05). Muscle HbO2 decreased while HHb increased during contractions with differences being observed among intensities (P < 0.05). Muscle Hbtot increased from rest at 20% MVC (P < 0.05), while no further change was observed at 40 and 60% MVC (P > 0.05). MCAv increased from rest to exercise but was not different among intensities (P > 0.05). Force output correlated with the root mean square EMG and changes in muscle HbO2 (P < 0.05), but not changes in cerebral HbO2 (P > 0.05) at all three intensities. Force output declined by 8% from the 1st to the 24th contraction only at 60% MVC and was accompanied by systematic increases in RMS, cerebral HbO2 and Hbtot with a leveling off in muscle HbO2 and Hbtot. These changes were independent of alterations in mean arterial pressure. Since cerebral blood flow and oxygenation were elevated at 60% MVC, we attribute the development of fatigue to reduced muscle oxygen availability rather than impaired central neuronal activation.

A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics.

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.

The barrel cortex as a model to study dynamic neuroglial interaction.

There is increasing evidence that glial cells, in particular astrocytes, interact dynamically with neurons. The well-known anatomofunctional organization of neurons in the barrel cortex offers a suitable and promising model to study such neuroglial interaction. This review summarizes and discusses recent in vitro as well as in vivo works demonstrating that astrocytes receive, integrate, and respond to neuronal signals. In addition, they are active elements of brain metabolism and exhibit a certain degree of plasticity that affects neuronal activity. Altogether these findings indicate that the barrel cortex presents glial compartments overlapping and interacting with neuronal compartments and that these properties help define barrels as functional and independent units. Finally, this review outlines how the use of the barrel cortex as a model might in the future help to address important questions related to dynamic neuroglia interaction.

Syndrome de vasoconstriction cérébrale réversible : identifications de facteurs pronostiques = [Reversible cerebral vasoconstriction syndrome identification of pronostic factors]

Objectif : Le syndrome de vasoconstriction cérébrale réversible (SVCR) est une entité clinico-radiologique associant des céphalées paroxystiques à un vasospasme uni- ou multifocal réversible des artères cérébrales avec ou sans déficit neurologique transitoire ou crise comitiale. Le but de notre étude est de rechercher les facteurs de mauvais pronostic des patients présentant un SVCR. Méthode : Nous avons réalisé une étude rétrospective des imageries vasculaires cérébrales invasives et non invasives entre janvier 2006 et 2011 et avons retenu 10 patients présentant les critères du RCVS. Les données démographiques, facteurs de risque vasculaires ainsi que l'évolution de chaque patient ont été noté. Résultats : Sept des 10 patients sont des femmes, avec un âge médian de 46 ans. Quatre patients ne présentaient pas de facteur étiologique, deux femmes se trouvaient en période post-partum (entre la première et la troisième semaine) et les trois autres cas sont induits par des drogues vaso-actives (cannabis pour 2 cas dont un associé à la cyclosporine, sumatriptan pour un cas). La durée moyenne du suivi est de 10,2 mois (0¬28 mois). Deux patients ont présentés une séquelle neurologique : un a gardé des troubles phasiques et l'autre une hémianopsie latérale homonyme. Deux autres patients sont décédés dans les suites, ce qui est inhabituel. Nous n'avons pas trouvé de corrélation d'évolution différente entre les cas de SVCR primaire ou secondaire. Les seules facteurs corrélaient à l'évolution clinique sont le status neurologique à l'admission et la présence de lésion parenchymateuse (ischémie ou hématome) à l'imagerie. Conclusion : La vasoconstriction cérébrale réversible impliquant des déficits neurologiques ou la mort a été, rarement, rapportée. Nous devons garder à l'esprit qu'une telle évolution peut survenir notamment pour les cas présentant un état neurologique dégradé à l'admission ou présentant des lésions parenchymateuses à l'imagerie.