Inflammation biomarkers in the intracranial blood are associated with outcome in patients with ischemic stroke.

BACKGROUND: Performing endovascular treatment (EVT) in patients with acute ischemic stroke (AIS) allows a port of entry for intracranial biological sampling. OBJECTIVE: To test the hypothesis that specific immune players are molecular contributors to disease, outcome biomarkers, and potential targets for modifying AIS. METHODS: We examined 75 subjects presenting with large vessel occlusion of the anterior circulation and undergoing EVT. Intracranial blood samples were obtained by microcatheter aspiration, as positioned for stent deployment. Peripheral blood samples were collected from the femoral artery. Plasma samples were quality controlled by electrophoresis and analyzed using a Mesoscale multiplex for targeted inflammatory and vascular factors. RESULTS: We measured 37 protein biomarkers in our sample cohort. Through multivariate analysis, adjusted for age, intravenous thrombolysis, pretreatment National Institutes of Health Stroke Scale and Alberta Stroke Program Early CT scores, we found that post-clot blood levels of interleukin-6 (IL-6) were significantly correlated (adjusted P value <0.05) with disability assessed by the modified Rankin Scale (mRS) score at 90 days, with medium effect size. Chemokine (C-C) ligand 17 CCL17/TARC levels were inversely correlated with the mRS score. Examination of peripheral blood showed that these correlations did not reach statistical significance after correction. Intracranial biomarker IL-6 level was specifically associated with a lower likelihood of favorable outcome, defined as a mRS score of 0-2. CONCLUSIONS: Our findings show a signature of blood inflammatory factors at the cerebrovascular occlusion site. The correlations between these acute-stage biomarkers and mRS score outcome support an avenue for add-on and localized immune modulatory strategies in AIS.

Continuous low-level dietary exposure to glyphosate elicits dose and sex-dependent synaptic and microglial adaptations in the rodent brain.

Prolonged exposure to low levels of dietary contaminants is a context in modern life that could alter organ physiology gradually. Here, we aimed to investigate the impact of continuous exposure to acceptable daily intake (ADI) and non-observable adverse effect level (NOAEL) of glyphosate from gestation to adulthood using C57BL/6J mice and incorporating these levels into their food pellets. From adulthood, we analyzed neurophysiological and neuro-glia cellular adaptations in male and female animals. Using ex-vivo hippocampal slice electrophysiology, we found a reduced efficacy of Schaffer collateral-to-CA1 excitatory synapses in glyphosate-exposed dietary conditions, with ADI and NOAEL dose-dependent effects. Short-term facilitation of excitatory synaptic transmission was specifically increased in NOAEL conditions, with a predominant influence in males, suggesting a reduced probability of neurotransmitter release. Long-term synaptic potentiation (LTP) was decreased in NOAEL-exposed mice. Next, we explore whether these neurophysiological modifications are associated with neuro-glia changes in the somatosensory cortex and hippocampus. High-resolution confocal microscopy analyses unveil a dose-dependent increased density of excitatory Vglut1+ Homer1+ synapses. Microglial Iba1+ cells displayed a shortening of their ramifications, a sign of cellular reactivity that was more pronounced in males at NOAEL levels. The morphology of GFAP+ astrocytes was generally not modified. Finally, we asked whether mouse-specific cross-correlations exist among all data sets generated. This examination included the novel object recognition (NOR) test performed before ex vivo functional and immunohistochemical examinations. We report a negative linear regression between the number of synapses and NOR or LTP maintenance when plotting ADI and NOAEL datasets. These results outline synaptic and microglial cell adaptations resulting from prenatal and continuous dietary low levels of glyphosate, discernible in, but not limited to, adult males exposed to the NOAEL. We discuss the potential significance of these findings to real-world consumer situations and long-term brain resilience.

Neurovascular multiparametric MRI defines epileptogenic and seizure propagation regions in experimental mesiotemporal lobe epilepsy.

OBJECTIVE: Improving the identification of the epileptogenic zone and associated seizure-spreading regions represents a significant challenge. Innovative brain-imaging modalities tracking neurovascular dynamics during seizures may provide new disease biomarkers. METHODS: With use of a multi-parametric magnetic resonance imaging (MRI) analysis at 9.4 Tesla, we examined, elaborated, and combined multiple cellular and cerebrovascular MRI read-outs as imaging biomarkers of the epileptogenic and seizure-propagating regions. Analyses were performed in an experimental model of mesial temporal lobe epilepsy (MTLE) generated by unilateral intra-hippocampal injection of kainic acid (KA). RESULTS: In the ipsilateral epileptogenic hippocampi, tissue T1 and blood-brain barrier (BBB) permeability to gadolinium were increased 48-72 hours post-KA, as compared to sham and contralateral hippocampi. BBB permeability endured during spontaneous focal seizures (4-6 weeks), along with a significant increase of apparent diffusion coefficient (ADC) and blood volume fraction (BVf). Simultaneously, ADC and BVf were augmented in the contralateral hippocampus, a region characterized by electroencephalographic seizure spreading, discrete histological neurovascular cell modifications, and no tissue sclerosis. We next asked whether combining all the acquired MRI parameters could deliver criteria to classify the epileptogenic from the seizure-spreading and sham hippocampi in these experimental conditions and over time. To differentiate sham from epileptogenic areas, the automatic multi-parametric classification provided a maximum accuracy of 97.5% (32 regions) 48-72 hours post-KA and of 100% (60 regions) at spontaneous seizures stage. To differentiate sham, epileptogenic, and seizure-spreading areas, the accuracies of the automatic classification were 93.1% (42 regions) 48-72 hours post-KA and 95% (80 regions) at spontaneous seizure stage. SIGNIFICANCE: Combining multi-parametric MRI acquisition and machine-learning analyses delivers specific imaging identifiers to segregate the epileptogenic from the contralateral seizure-spreading hippocampi in experimental MTLE. The potential clinical value of our findings is critically discussed.

Varying modalities of perinatal exposure to a pesticide cocktail elicit neurological adaptations in mice and zebrafish.

Epidemiological indications connect maternal and developmental presence or exposure to pesticides with an increased risk for a spectrum of neurological trajectories. To provide pre-clinical data in support of this hypothesis, we used two distinct experimental models. First, female and male mice were fed immediately prior to mating, and the resulting pregnant dams were continously fed during gestation and lactation periods using chow pellets containing a cocktail of six pesticides at tolerable daily intake levels. Male and female offspring were then tracked for behavioral and in vivo electrophysiological adaptations. Second, a zebrafish model allowed us to screen toxicity and motor-behavior outcomes specifically associated with the developmental exposure to a low-to-high concentration range of the cocktail and of each individual pesticide. Here, we report anxiety-like behavior in aging male mice maternally exposed to the cocktail, as compared to age and gender matched sham animals. In parallel, in vivo electrocorticography revealed a decrease in gamma (40-80 Hz) and an increase of theta (6-9 Hz) waves, delineating a long-term, age-dependent, neuronal slowing. Neurological changes were not accompanied by brain structural malformations. Next, by using zebrafish larvae, we showed an increase of all motor-behavioral parameters resulting from the developmental exposure to 10 µg/L of pesticide cocktail, an outcome that was not associated with midbrain structural or neurovascular modifications as assessed by in vivo 2-photon microscopy. When screening each pesticide, chlorpyrifos elicited modifications of swimming parameters at 0.1 µg/L, while other components provoked changes from 0.5 µg/L. Ziram was the single most toxic component inducing developmental malformations and mortality at 10 µg/L. Although we have employed non-equivalent modalities and timing of exposure in two dissimilar experimental models, these outcomes indicate that presence of a pesticide cocktail during perinatal periods represents an element promoting behavioral and neurophysiological modifications. The study limitations and the possible pertinence of our findings to ecotoxicology and public health are critically discussed.

Differential impact of dose-range glyphosate on locomotor behavior, neuronal activity, glio-cerebrovascular structures, and transcript regulations in zebrafish larvae.

The presence of glyphosate represents a debated ecotoxicological and health risk factor. Here, zebrafish larvae were exposed, from 1.5 to 120 h post-fertilization, to a broad concentration range (0.05-10.000 µg/L) of glyphosate to explore its impact on the brain. We evaluated morphology, tracked locomotor behavior and neurophysiological parameters, examined neuro-glio-vascular cell structures, and outlined transcriptomic outcomes by RNA sequencing. At the concentration range tested, glyphosate did not elicit gross morphological changes. Behavioral analysis revealed a significant decrease in locomotor activity following the exposure to 1000 µg/L glyphosate or higher. In parallel, midbrain electrophysiological recordings indicated abnormal, and variable, spike activity in zebrafish larvae exposed to 1000 µg/L glyphosate. Next, we asked whether the observed neurophysiological outcome could be secondary to brain structural modifications. We used transgenic zebrafish and in vivo 2-photon microscopy to examine, at the cellular level, the effects of the behavior-modifying concentration of 1000 µg/L, comparing to low 0.1 µg/L, and control. We ruled out the presence of cerebrovascular and neuronal malformations. However, microglia morphological modifications were visible at the two glyphosate concentrations, specifically the presence of amoeboid cells suggestive of activation. Lastly, RNAseq analysis showed the deregulation of transcript families implicated in neuronal physiology, synaptic transmission, and inflammation, as evaluated at the two selected glyphosate concentrations. In zebrafish larvae, behavioral and neurophysiological defects occur after the exposure to high glyphosate concentrations while cellular and transcript signatures can be detected in response to low dose. The prospective applicability to ecotoxicology and the possible extension to brain-health vulnerability are critically discussed.

Machine Learning Analysis of the Cerebrovascular Thrombi Proteome in Human Ischemic Stroke: An Exploratory Study.

Objective: Mechanical retrieval of thrombotic material from acute ischemic stroke patients provides a unique entry point for translational research investigations. Here, we resolved the proteomes of cardioembolic and atherothrombotic cerebrovascular human thrombi and applied an artificial intelligence routine to examine protein signatures between the two selected groups. Methods: We specifically used n = 32 cardioembolic and n = 28 atherothrombotic diagnosed thrombi from patients suffering from acute stroke and treated by mechanical thrombectomy. Thrombi proteins were successfully separated by gel-electrophoresis. For each thrombi, peptide samples were analyzed by nano-flow liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) to obtain specific proteomes. Relative protein quantification was performed using a label-free LFQ algorithm and all dataset were analyzed using a support-vector-machine (SVM) learning method. Data are available via ProteomeXchange with identifier PXD020398. Clinical data were also analyzed using SVM, alone or in combination with the proteomes. Results: A total of 2,455 proteins were identified by nano-LC-MS/MS in the samples analyzed, with 438 proteins constantly detected in all samples. SVM analysis of LFQ proteomic data delivered combinations of three proteins achieving a maximum of 88.3% for correct classification of the cardioembolic and atherothrombotic samples in our cohort. The coagulation factor XIII appeared in all of the SVM protein trios, associating with cardioembolic thrombi. A combined SVM analysis of the LFQ proteome and clinical data did not deliver a better discriminatory score as compared to the proteome only. Conclusion: Our results advance the portrayal of the human cerebrovascular thrombi proteome. The exploratory SVM analysis outlined sets of proteins for a proof-of-principle characterization of our cohort cardioembolic and atherothrombotic samples. The integrated analysis proposed herein could be further developed and retested on a larger patients population to better understand stroke origin and the associated cerebrovascular pathophysiology.

Zika Virus Infection Promotes Local Inflammation, Cell Adhesion Molecule Upregulation, and Leukocyte Recruitment at the Blood-Brain Barrier.

The blood-brain barrier (BBB) largely prevents toxins and pathogens from accessing the brain. Some viruses have the ability to cross this barrier and replicate in the central nervous system (CNS). Zika virus (ZIKV) was responsible in 2015 to 2016 for a major epidemic in South America and was associated in some cases with neurological impairments. Here, we characterized some of the mechanisms behind its neuroinvasion using an innovative in vitro human BBB model. ZIKV efficiently replicated, was released on the BBB parenchyma side, and triggered subtle modulation of BBB integrity as well as an upregulation of inflammatory and cell adhesion molecules (CAMs), which in turn favored leukocyte recruitment. Finally, we showed that ZIKV-infected mouse models displayed similar CAM upregulation and that soluble CAMs were increased in plasma samples from ZIKV-infected patients. Our observations suggest a complex interplay between ZIKV and the BBB, which may trigger local inflammation, leukocyte recruitment, and possible cerebral vasculature impairment.IMPORTANCE Zika virus (ZIKV) can be associated with neurological impairment in children and adults. To reach the central nervous system, viruses have to cross the blood-brain barrier (BBB), a multicellular system allowing a tight separation between the bloodstream and the brain. Here, we show that ZIKV infects cells of the BBB and triggers a subtle change in its permeability. Moreover, ZIKV infection leads to the production of inflammatory molecules known to modulate BBB integrity and participate in immune cell attraction. The virus also led to the upregulation of cellular adhesion molecules (CAMs), which in turn favored immune cell binding to the BBB and potentially increased infiltration into the brain. These results were also observed in a mouse model of ZIKV infection. Furthermore, plasma samples from ZIKV-infected patients displayed an increase in CAMs, suggesting that this mechanism could be involved in neuroinflammation triggered by ZIKV.

Perinatal exposure to a dietary pesticide cocktail does not increase susceptibility to high-fat diet-induced metabolic perturbations at adulthood but modifies urinary and fecal metabolic fingerprints in C57Bl6/J mice.

BACKGROUND: We recently demonstrated that chronic dietary exposure to a mixture of pesticides at low-doses induced sexually dimorphic obesogenic and diabetogenic effects in adult mice. Perinatal pesticide exposure may also be a factor in metabolic disease etiology. However, the long-term consequences of perinatal pesticide exposure remain controversial and largely unexplored. OBJECTIVES: Here we assessed how perinatal exposure to the same low-dose pesticide cocktail impacted metabolic homeostasis in adult mice. METHODS: Six pesticides (boscalid, captan, chlopyrifos, thiachloprid, thiophanate, and ziram) were incorporated in food pellets. During the gestation and lactation periods, female (F0) mice were fed either a pesticide-free or a pesticide-enriched diet at doses exposing them to the tolerable daily intake (TDI) level for each compound, using a 1:1 body weight scaling from humans to mice. All male and female offsprings (F1) were then fed the pesticide-free diet until 18 weeks of age, followed by challenge with a pesticide-free high-fat diet (HFD) for 6 weeks. Metabolic parameters, including body weight, food and water consumption, glucose tolerance, and urinary and fecal metabolomes, were assessed over time. At the end of the experiment, we evaluated energetic metabolism and microbiota activity using biochemical assays, gene expression profiling, and 1H NMR-based metabolomics in the liver, urine, and feces. RESULTS: Perinatal pesticide exposure did not affect body weight or energy homeostasis in 6- and 14-week-old mice. As expected, HFD increased body weight and induced metabolic disorders as compared to a low-fat diet. However, HFD-induced metabolic perturbations were similar between mice with and without perinatal pesticide exposure. Interestingly, perinatal pesticide exposure induced time-specific and sex-specific alterations in the urinary and fecal metabolomes of adult mice, suggesting long-lasting changes in gut microbiota. CONCLUSIONS: Perinatal pesticide exposure induced sustained sexually dimorphic perturbations of the urinary and fecal metabolic fingerprints, but did not significantly influence the development of HFD-induced metabolic diseases.

Life-long Dietary Pesticide Cocktail Induces Astrogliosis Along with Behavioral Adaptations and Activates p450 Metabolic Pathways.

Exposure to environmental contaminants is a public health concern. However, pre-clinical studies that examine the impact of pesticides at low-dose and the long-term consequences are uncommon. Here, C57BL6/j male and female mice were daily fed from weaning and up to 12 months, corresponding to early-childhood into middle-age in humans, using chow pellets containing a cocktail of pesticides at tolerable daily intake levels. We found that 12 months of dietary exposure to pesticides was associated with a moderate perenchymal or perivascular astrogliosis in specific hippocampal sub-regions. The expression of platelet-derived growth factor receptor beta was modified at the perivascular level. Examination of Iba1+ microglial cells did not reveal sizeable changes. Concomitantly to astrogliosis, spontaneous spatial memory and sociability were modified in males at 12 months of dietary exposure to pesticides. Telemetry electrocorticograhic explorations ruled out the presence of epileptiform activity or theta-gamma wave modifications in these conditions. Long-term pesticides impacted the periphery where the hepatic P450 metabolic cytochromes Cyp4a14 and Cyp4a10 were significantly upregulated in male and female mice during the 12 months of exposure. The expression of ß-oxidation genes, such as Acox1, Cpt1a and Eci, was also significantly increased in male and female mice in response to pesticides. Collectively, our results indicate that a life-long exposure to a pesticide cocktail elicits sex-dependent, spatio-temporally restricted brain modifications and significant activation of P450 pathways in the periphery. These brain-peripheral adjustments are discussed as time or age-dependent vulnerability elements.

The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.

Immune changes occur in experimental and clinical epilepsy. Here, we tested the hypothesis that during epileptogenesis and spontaneous recurrent seizures (SRS) an impairment of the endogenous anti-inflammatory pathway glucocorticoid receptor (GR)-annexin A1 (ANXA1) occurs. By administrating exogenous ANXA1, we studied whether pharmacological potentiation of the anti-inflammatory response modifies seizure activity and pathophysiology. We used an in vivo model of temporal lobe epilepsy based on intrahippocampal kainic acid (KA) injection. Video-electroencephalography, molecular biology analyses on brain and peripheral blood samples, and pharmacological investigations were performed in this model. Human epileptic cortices presenting type II focal cortical dysplasia (IIa and b), hippocampi with or without hippocampal sclerosis (HS), and available controls were used to study ANXA1 expression. A decrease of phosphorylated (phospho-) GR and phospho-GR/tot-GR protein expression occurred in the hippocampus during epileptogenesis. Downstream to GR, the anti-inflammatory protein ANXA1 remained at baseline levels while inflammation installed and endured. In peripheral blood, ANXA1 and corticosterone levels showed no significant modifications during disease progression except for an early and transient increase poststatus epilepticus. These results indicate inadequate ANXA1 engagement over time and in these experimental conditions. By analyzing human brain specimens, we found that where significant inflammation exists, the pattern of ANXA1 immunoreactivity was abnormal because the typical perivascular ANXA1 immunoreactivity was reduced. We next asked whether potentiation of the endogenous anti-inflammatory mechanism by ANXA1 administration modifies the disease pathophysiology. Although with varying efficacy, administration of exogenous ANXA1 somewhat reduced the time spent in seizure activity as compared to saline. These results indicate that the anti-inflammatory GR-ANXA1 pathway is defective during experimental seizure progression. The prospect of pharmacologically restoring or potentiating this endogenous anti-inflammatory mechanism as an add-on therapeutic strategy for specific forms of epilepsy is proposed.-Zub, E., Canet, G., Garbelli, R., Blaquiere, M., Rossini, L., Pastori, C., Sheikh, M., Reutelingsperger, C., Klement, W., de Bock, F., Audinat, E., Givalois, L., Solito, E., Marchi, N. The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.

Seizure progression and inflammatory mediators promote pericytosis and pericyte-microglia clustering at the cerebrovasculature.

BACKGROUND: Cerebrovascular dysfunction and inflammation occur in epilepsy. Here we asked whether pericytes, a pivotal cellular component of brain capillaries, undergo pathological modifications during experimental epileptogenesis and in human epilepsy. We evaluated whether pro-inflammatory cytokines, present in the brain during seizures, contribute to pericyte morphological modifications. METHODS: In vivo, unilateral intra-hippocampal kainic acid (KA) injections were performed in NG2DsRed/C57BL6 mice to induce status epilepticus (SE), epileptogenesis, and spontaneous recurrent seizures (SRS). NG2DsRed mice were used to visualize pericytes during seizure progression. The effect triggered by recombinant IL-1ß, TNFα, or IL-6 on pericytes was evaluated in NG2DsRed hippocampal slices and in human-derived cell culture. Human brain specimens obtained from temporal lobe epilepsy (TLE) with or without sclerosis (HS) and focal cortical dysplasia (FCD-IIb) were evaluated for pericyte-microglial cerebrovascular assembly. RESULTS: A disarray of NG2DsRed+ pericyte soma and ramifications was found 72 h post-SE and 1 week post-SE (epileptogenesis) in the hippocampus. Pericyte modifications topographically overlapped with IBA1+ microglia clustering around the capillaries with cases of pericytes lodged within the microglial cells. Microglial clustering around the NG2DsRed pericytes lingered at SRS. Pericyte proliferation (Ki67+) occurred 72 h post-SE and during epileptogenesis and returned towards control levels at SRS. Human epileptic brain tissues showed pericyte-microglia assemblies with IBA1/HLA microglial cells outlining the capillary wall in TLE-HS and FCD-IIb specimens. Inflammatory mediators contributed to pericyte modifications, in particular IL-1ß elicited pericyte morphological changes and pericyte-microglia clustering in NG2DsRed hippocampal slices. Modifications also occurred when pro-inflammatory cytokines were added to an in vitro culture of pericytes. CONCLUSIONS: These results indicate the occurrence of pericytosis during seizures and introduce a pericyte-microglial mediated mechanism of blood-brain barrier dysfunction in epilepsy.

Cerebrovascular heterogeneity and neuronal excitability.

The cerebral vasculature is a complex tridimensional network of arterial and venous vessels which are anatomically in proximity of and functionally coupled to neurons. Depending on the cellular composition of the vascular wall and size, cerebral vessels control regional blood flow, define interstitial homeostasis or cerebrospinal fluid circulation and influence immune cell patrolling. Pathological deviations from these functions promote or are a consequence of brain diseases, directly impacting neuronal firing. We propose that specific cerebrovascular segments are differentially implicated in the pathophysiology of epilepsy, including difference between white and grey matter. We offer plasticity of perivascular mural cells and endothelial-pericyte interactions as emerging players. We outline the potential for MRI vascular biomarkers tailored to the epileptic brain, specifically cerebral blood volume and flow, tissue oxygen saturation and microvessel permeability. Finally, we show the advantages of the guinea pig whole brain preparation to study the link between cerebrovascular permeability, expression of vascular adhesion molecules, inflammation and neuronal excitability.

Lack of CAR impacts neuronal function and cerebrovascular integrity in vivo.

Nuclear receptors (NRs) are a group of transcription factors emerging as players in normal and pathological CNS development. Clinically, an association between the constitutive androstane NR (CAR) and cognitive impairment was proposed, however never experimentally investigated. We wished to test the hypothesis that the impact of CAR on neurophysiology and behavior is underlined by cerebrovascular-neuronal modifications. We have used CAR(-/-) C57BL/6 and wild type mice and performed a battery of behavioral tests (recognition, memory, motor coordination, learning and anxiety) as well as longitudinal video-electroencephalographic recordings (EEG). Brain cell morphology was assessed using 2-photon or electron microscopy and fluorescent immunohistochemistry. We observed recognition memory impairment and increased anxiety-like behavior in CAR(-/-) mice, while locomotor activity was not affected. Concomitantly to memory deficits, EEG monitoring revealed a decrease in 3.5-7Hz waves during the awake/exploration and sleep periods. Behavioral and EEG abnormalities in CAR(-/-) mice mirrored structural changes, including tortuous fronto-parietal penetrating vessels. At the cellular level we found reduced ZO-1, but not CLDN5, tight junction protein expression in cortical and hippocampal isolated microvessel preparations. Interestingly, the neurotoxin kainic acid, when injected peripherally, provoked a rapid onset of generalized convulsions in CAR(-/-) as compared to WT mice, supporting the hypothesis of vascular permeability. The morphological phenotype of CAR(-/-) mice also included some modifications of GFAP/IBA1 glial cells in the parenchymal or adjacent to collagen-IV(+) or FITC(+) microvessels. Neuronal defects were also observed including increased cortical NEUN(+) cell density, hippocampal granule cell dispersion and increased NPY immunoreactivity in the CA1 region in CAR(-/-) mice. The latter may contribute to the in vivo phenotype. Our results indicate that behavioral and electroencephalographic changes in adult CAR(-/-) mice are concomitant to discrete developmental or structural brain defects. The latter could increase the vulnerability to neurotoxins. The possibility that interfering with nuclear receptors during development could contribute to adulthood brain changes is proposed.

Redistribution of PDGFRß cells and NG2DsRed pericytes at the cerebrovasculature after status epilepticus.

PURPOSE: The role of cerebrovascular dysfunction in seizure disorders is recognized. Blood-brain barrier (BBB) damage in epilepsy has been linked to endothelial and glial pathophysiological changes. Little is known about the involvement of pericytes, a cell type that contributes to BBB function. METHODS: NG2DsRed mice were used to visualize cerebrovascular pericytes. The pattern of vascular and parenchymal distributions of platelet-derived growth factor receptor beta (PDGFRß) cells was evaluated by immunohistochemistry. Status epilepticus was induced in NG2DsRed or C57BL/6J mice by intraperitoneal kainic acid (KA). Animals were perfused intracardially using FITC-Dextran or FITC-Albumin to visualize the cerebrovasculature. Colocalization was performed between NG2DsRed, PDGFRß and microglia IBA-1. Confocal 3D vessel reconstruction was used to visualize changes in cell morphology and position. PDGFRß expression was also evaluated in vitro using organotypic hippocampal cultures (OHC) treated with kainic acid to induce seizure-like activity. Co-localization of PDGFRß with the vascular marker RECA-1 and NG2 was performed. Finally, we assessed the expression of PDGFRß in brain specimens obtained from a cohort of patients affected by drug resistant epilepsy compared to available autoptic brain. RESULTS: In vivo, severe status epilepticus (SE) altered NG2DsRed vascular coverage. We found dishomogenous NG2DsRed perivascular ramifications after SE and compared to control. Concomitantly, PDGFRß(+) cells re-distributed towards the cerebrovasculature after severe SE. Cerebrovascular NG2DsRed partially colocalized with PDGFRß(+) while parenchymal PDGFRß(+) cells did not colocalize with IBA-1(+) microglia. Using in vitro OHC we found decreased NG2 vascular staining and increased PDGFRß(+) ramifications associated with RECA-1(+) microvessels after seizure-like activity. Cellular PDGFRß and NG2(+) colocalization was observed in the parenchyma. Finally, analysis of human TLE brains revealed perivascular and parenchymal PDGFRß(+) cell distributions resembling the murine in vivo and in vitro results. PDGFRß(+) cells at the cerebrovasculature were more frequent in TLE brain tissues as compared to the autoptic control. CONCLUSIONS: The rearrangement of PDGFRß(+) and vascular NG2DsRed cells after SE suggests a possible involvement of pericytes in the cerebrovascular modifications observed in epilepsy. The functional role of vascular-parenchymal PDGFRß(+) cell redistribution and the relevance of a pericyte response to SE remain to be fully elucidated.

A presynaptic role of microtubule-associated protein 1/Futsch in Drosophila: regulation of active zone number and neurotransmitter release.

Structural microtubule-associated proteins (MAPs), like MAP1, not only control the stability of microtubules, but also interact with postsynaptic proteins in the nervous system. Their presynaptic role has barely been studied. To tackle this question, we used the Drosophila model in which there is only one MAP1 homolog: Futsch, which is expressed at the larval neuromuscular junction, presynaptically only. We show that Futsch regulates neurotransmitter release and active zone density. Importantly, we provide evidence that this role of Futsch is not just the consequence of its microtubule-stabilizing function. Using high-resolution microscopy, we show that Futsch and microtubules are almost systematically present in close proximity to active zones, with Futsch being localized in-between microtubules and active zones. Using proximity ligation assays, we further demonstrate the proximity of Futsch, but not microtubules, to active zone components. Altogether our data are in favor of a model by which Futsch locally stabilizes active zones, by reinforcing their link with the underlying microtubule cytoskeleton.

Organotypic brain slices: a model to study the neurovascular unit micro-environment in epilepsies.

BACKGROUND: It is now recognized that the neuro-vascular unit (NVU) plays a key role in several neurological diseases including epilepsy, stroke, Alzheimer's disease, multiple sclerosis and the development of gliomas. Most of these disorders are associated with NVU dysfunction, due to overexpression of inflammatory factors such as vascular endothelial growth factor (VEGF). Various in vitro models have been developed previously to study the micro-environment of the blood-brain barrier (BBB). However none of these in vitro models contained a complete complement of NVU cells, nor maintained their interactions, thus minimizing the influence of the surrounding tissue on the BBB development and function. The organotypic hippocampal culture (OHC) is an integrative in vitro model that allows repeated manipulations over time to further understand the development of cell circuits or the mechanisms of brain diseases. METHODS/DESIGN: OHCs were cultured from hippocampi of 6-7 day-old Sprague Dawley rats. After 2 weeks in culture, seizures were induced by application of kainate or bicuculline into culture medium. The regulation of BBB integrity under physiological and pathological conditions was evaluated by immunostaining of the main tight junction (TJ) proteins and of the basal membrane of microvessels. To mimic or prevent BBB disassembly, we used diverse pro- or anti-angiogenic treatments. DISCUSSION: This study demonstrates that NVU regulation can be investigated using OHCs. We observed in this model system an increase in vascularization and a down-regulation of TJ proteins, similar to the vascular changes described in a chronic focus of epileptic patients, and in rodent models of epilepsy or inflammation. We observed that Zonula occludens-1 (ZO-1) protein disappeared after seizures associated with neuronal damage. In these conditions, the angiopoeitin-1 system was down-regulated, and the application of r-angiopoeitin-1 allowed TJ re-assembly. This article demonstrates that organotypic culture is a useful model to decipher the links between epileptic activity and vascular damage, and also to investigate NVU regulation in diverse neurological disorders.

Epileptiform activity induces vascular remodeling and zonula occludens 1 downregulation in organotypic hippocampal cultures: role of VEGF signaling pathways.

Recent studies suggest that blood-brain barrier (BBB) permeability contributes to epileptogenesis in symptomatic epilepsies. We have previously described angiogenesis, aberrant vascularization, and BBB alteration in drug-refractory temporal lobe epilepsy. Here, we investigated the role of vascular endothelial growth factor (VEGF) in an in vitro integrative model of vascular remodeling induced by epileptiform activity in rat organotypic hippocampal cultures. After kainate-induced seizure-like events (SLEs), we observed an overexpression of VEGF and VEGF receptor-2 (VEGFR-2) as well as receptor activation. Vascular density and branching were significantly increased, whereas zonula occludens 1 (ZO-1), a key protein of tight junctions (TJs), was downregulated. These effects were fully prevented by VEGF neutralization. Using selective inhibitors of VEGFR-2 signaling pathways, we found that phosphatidylinositol 3-kinase is involved in cell survival, protein kinase C (PKC) in vascularization, and Src in ZO-1 regulation. Recombinant VEGF reproduced the kainate-induced vascular changes. As in the kainate model, VEGFR-2 and Src were involved in ZO-1 downregulation. These results showed that VEGF/VEGFR-2 initiates the vascular remodeling induced by SLEs and pointed out the roles of PKC in vascularization and Src in TJ dysfunction, respectively. This suggests that Src pathway could be a therapeutic target for BBB protection in epilepsies.

Epac mediates PACAP-dependent long-term depression in the hippocampus.

Extensive work has shown that activation of the cAMP-dependent protein kinase A (PKA) is crucial for long-term depression (LTD) of synaptic transmission in the hippocampus, a phenomenon that is thought to be involved in memory formation. Here we studied the role of an alternative target of cAMP, the exchange protein factor directly activated by cyclic AMP (Epac). We show that pharmacological activation of Epac by the selective agonist 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT) induces LTD in the CA1 region. Paired-pulse facilitation of synaptic responses remained unchanged after induction of this LTD, suggesting that it depended on postsynaptic mechanisms. The 8-pCPT-induced LTD was blocked by the Epac signalling inhibitor brefeldin-A (BFA), Rap-1 antagonist geranylgeranyltransferase inhibitor (GGTI) and p38 mitogen activated protein kinase (P38-MAPK) inhibitor SB203580. This indicated a direct involvement of Epac in this form of LTD. As for other forms of LTD, a mimetic peptide of the PSD-95/Disc-large/ZO-1 homology (PDZ) ligand motif of the AMPA receptor subunit GluR2 blocked the Epac-LTD, suggesting involvement of PDZ protein interaction. The Epac-LTD also depended on mobilization of intracellular Ca(2+), proteasome activity and mRNA translation, but not transcription, as it was inhibited by thapsigargin, lactacystin and anisomycin, but not actinomycin-D, respectively. Finally, we found that the pituitary adenylate cyclase activating polypeptide (PACAP) can induce an LTD that was mutually occluded by the Epac-LTD and blocked by BFA or SB203580, suggesting that the Epac-LTD could be mobilized by stimulation of PACAP receptors. Altogether these results provided evidence for a new form of hippocampal LTD.

PICK1 uncoupling from mGluR7a causes absence-like seizures.

Absence epilepsy is a neurological disorder that causes a recurrent loss of consciousness and generalized spike-and-wave discharges on an electroencephalogram (EEG). The role of metabotropic glutamate receptors (mGluRs) and associated scaffolding proteins in absence epilepsy has been unclear to date. We investigated a possible role for these proteins in absence epilepsy, focusing on the mGluR7a receptor and its PDZ-interacting protein, protein interacting with C kinase 1 (PICK1), in rats and mice. Injection of a cell-permeant dominant-negative peptide or targeted mutation of the mGluR7a C terminus, both of which disrupt the interaction between the receptor and PDZ proteins, caused behavioral symptoms and EEG discharges that are characteristic of absence epilepsy. Inactivation of the Pick1 gene also facilitated pharmacological induction of the absence epilepsy phenotype. The cortex and thalamus, which are known to participate in absence epilepsy, were involved, but the hippocampus was not. Our results indicate that disruption of the mGluR7a-PICK1 complex is sufficient to induce absence epilepsy-like seizures in rats and mice, thus providing, to the best of our knowledge, the first animal model of metabotropic glutamate receptor-PDZ protein interaction in absence epilepsy.

Loss of X-linked mental retardation gene oligophrenin1 in mice impairs spatial memory and leads to ventricular enlargement and dendritic spine immaturity.

Loss of oligophrenin1 (OPHN1) function in human causes X-linked mental retardation associated with cerebellar hypoplasia and, in some cases, with lateral ventricle enlargement. In vitro studies showed that ophn1 regulates dendritic spine through the control of Rho GTPases, but its in vivo function remains unknown. We generated a mouse model of ophn1 deficiency and showed that it mimics the ventricles enlargement without affecting the cerebellum morphoanatomy. The ophn1 knock-out mice exhibit behavioral defects in spatial memory together with impairment in social behavior, lateralization, and hyperactivity. Long-term potentiation and mGluR-dependent long-term depression are normal in the CA1 hippocampal area of ophn1 mutant, whereas paired-pulse facilitation is reduced. This altered short-term plasticity that reflects changes in the release of neurotransmitters from the presynaptic processes is associated with normal synaptic density together with a reduction in mature dendritic spines. In culture, inactivation of ophn1 function increases the density and proportion of immature spines. Using a conditional model of loss of ophn1 function, we confirmed this immaturity defect and showed that ophn1 is required at all the stages of the development. These studies show that, depending of the context, ophn1 controls the maturation of dendritic spines either by maintaining the density of mature spines or by limiting the extension of new filopodia. Altogether, these observations indicate that cognitive impairment related to OPHN1 loss of function is associated with both presynaptic and postsynaptic alterations.