Epileptiform activity contralateral to unilateral hippocampal sclerosis does not cause the expression of brain damage markers.

OBJECTIVE: Patients with epilepsy often ask if recurrent seizures harm their brain and aggravate their epileptic condition. This crucial question has not been specifically addressed by dedicated experiments. We analyze here if intense bilateral seizure activity induced by local injection of kainic acid (KA) in the right hippocampus produces brain damage in the left hippocampus. METHODS: Adult guinea pigs were bilaterally implanted with hippocampal electrodes for continuous video-electroencephalography (EEG) monitoring. Unilateral injection of 1 µg KA in the dorsal CA1 area induced nonconvulsive status epilepticus (ncSE) characterized by bilateral hippocampal seizure discharges. This treatment resulted in selective unilateral sclerosis of the KA-injected hippocampus. Three days after KA injection, the animals were killed, and the brains were submitted to ex vivo magnetic resonance imaging (MRI) and were processed for immunohistochemical analysis. RESULTS: During ncSE, epileptiform activity was recorded for 27.6 ± 19.1 hours in both the KA-injected and contralateral hippocampi. Enhanced T1-weighted MR signal due to gadolinium deposition, mean diffusivity reduction, neuronal loss, gliosis, and blood-brain barrier permeability changes was observed exclusively in the KA-injected hippocampus. Despite the presence of a clear unilateral hippocampal sclerosis at the site of KA injection, no structural alterations were detected by MR and immunostaining analysis performed in the hippocampus contralateral to KA injection 3 days and 2 months after ncSE induction. Fluoro-Jade and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at the same time points confirmed the absence of degenerating cells in the hippocampi contralateral to KA injection. SIGNIFICANCE: We demonstrate that intense epileptiform activity during ncSE does not cause obvious brain damage in the hippocampus contralateral to unilateral hippocampal KA injection. These findings argue against the hypothesis that epileptiform activity per se contributes to focal brain injury in previously undamaged cortical regions.

Kainic acid-induced albumin leak across the blood-brain barrier facilitates epileptiform hyperexcitability in limbic regions.

OBJECTIVE: Systemic administration of kainic acid (KA) is a widely used procedure utilized to develop a model of temporal lobe epilepsy (TLE). Despite its ability to induce status epilepticus (SE) in vivo, KA applied to in vitro preparations induces only interictal-like activity and/or isolated ictal discharges. The possibility that extravasation of the serum protein albumin from the vascular compartment enhances KA-induced brain excitability is investigated here. METHODS: Epileptiform activity was induced by arterial perfusion of 6 µm KA in the in vitro isolated guinea pig brain preparation. Simultaneous field potential recordings were carried out bilaterally from limbic (CA1, dentate gyrus [DG], and entorhinal cortex) and extralimbic regions (piriform cortex and neocortex). Blood-brain barrier (BBB) breakdown associated with KA-induced epileptiform activity was assessed by parenchymal leakage of intravascular fluorescein-isothiocyanate albumin. Seizure-induced brain inflammation was evaluated by western blot analysis of interleukin (IL)-1ß expression in brain tissue. RESULTS: KA infusion caused synchronized activity at 15-30 Hz in limbic (but not extralimbic) cortical areas, associated with a brief, single seizure-like event. A second bolus of KA, 60 min after the induction of the first ictal event, did not further enhance excitability. Perfusion of serum albumin between the two administrations of KA enhanced epileptiform discharges and allowed a recurrent ictal event during the second KA infusion. SIGNIFICANCE: Our data show that arterial KA administration selectively alters the synchronization of limbic networks. However, KA is not sufficient to generate recurrent seizures unless serum albumin is co-perfused during KA administration. These findings suggest a role of serum albumin in facilitating acute seizure generation.

Increased pCREB expression and the spontaneous epileptiform activity in a BCNU-treated rat model of cortical dysplasia.

OBJECTIVE: Cortical dysplasias (CDs) represent a wide range of cortical abnormalities that closely correlate with intractable epilepsy. Rats prenatally exposed to 1-3-bis-chloroethyl-nitrosurea (BCNU) represent an injury-based model that reproduces many histopathologic features of human CD. Previous studies reported in vivo hyperexcitability in this model, but in vivo epileptogenicity has not been confirmed. METHODS: To determine whether cortical and hippocampal lesions lead to epileptiform discharges and/or seizures in the BCNU model, rats at three different ages (3, 5, and 9 months old) were implanted for long-term video electroencephalographic recording. At the end of the recording session, brain tissue was processed for histologic and immunohistochemical investigation including cAMP response element binding protein (CREB) phosphorylation, as a biomarker of epileptogenicity. RESULTS: BCNU-treated rats showed spontaneous epileptiform activity (67%) in the absence of a second seizure-provoking hit. Such activity originated mainly from one hippocampus and propagated to the ipsilateral neocortex. No epileptiform activity was found in age-matched control rats. The histopathologic investigation revealed that all BCNU rats with epileptiform activity showed neocortical and hippocampal abnormalities; the presence and the severity of these lesions did not correlate consistently with the propensity to generate epileptiform discharges. Epileptiform activity was found only in cortical areas of BCNU-treated rats in which a correlation between brain abnormalities and increased pCREB expression was observed. SIGNIFICANCE: This study demonstrates the in vivo occurrence of spontaneous epileptiform discharges in the BCNU model and shows that increased pCREB expression can be utilized as a reliable biomarker of epileptogenicity.

Variable electrobehavioral patterns during focal nonconvulsive status epilepticus induced by unilateral intrahippocampal injection of kainic acid.

OBJECTIVE: Nonconvulsive status epilepticus (ncSE) is a severe condition that may result in neurologic sequelae and epilepsy resistant to pharmacologic treatment. We analyze here seizure and electroencephalography (EEG) patterns and their correlation to the development of a chronic epileptic condition in a guinea pig model of focal ncSE induced by intrahippocampal injection of kainic acid (KA). METHODS: Electrobehavioral patterns during ncSE induced by unilateral injection of 1 µg of KA in the CA1 region of the hippocampus were characterized by continuous video-EEG monitoring in 13 guinea pigs bilaterally implanted with recording electrodes in the hippocampus and neocortex. RESULTS: Video-EEG analysis demonstrates a high variability of seizure type and duration during KA-induced ncSE. Seizures showed focal signs correlated with diverse epileptiform EEG discharge distributions, either diffuse or localized. Nonfocal (bilateral motor) signs during seizures most likely correlated with a diffuse EEG pattern. The evolution into a chronic epileptic condition correlated neither with the severity of seizure pattern nor with the diffusion of the EEG discharges observed during the ncSE. SIGNIFICANCE: Video-EEG monitoring in a guinea pig model of ncSE induced by unilateral hippocampal injection of KA demonstrates a high variability of electrobehavioral patterns. We demonstrate that the seizure severity score during focal ncSE is not a predictor of the evolution into a chronic epileptic condition of mesial temporal lobe epilepsy.

Seizure-induced brain-borne inflammation sustains seizure recurrence and blood-brain barrier damage.

OBJECTIVE: Epilepsy is a common neurological disorder characterized by recurrent seizures often unresponsive to pharmacological treatment. Brain inflammation is considered a crucial etiopathogenetic mechanism of epilepsy that could be targeted to control seizures. Specific inflammatory mediators overexpressed in human epileptogenic foci are known to promote seizures in animal models. We investigated whether seizures induce brain inflammation independently on extracerebral factors. We also investigated whether brain-borne inflammation is required and sufficient to maintain seizure activity and whether it causes blood-brain barrier (BBB) impairment. We addressed these questions by studying the relation between seizures, inflammation, and BBB permeability in a brain preparation isolated from extracerebral compartments. METHODS: Epileptiform activity was induced by arterial perfusion of bicuculline in the in vitro isolated guinea pig brain. Seizure-induced brain inflammation was evaluated by quantitative immunohistochemical analysis of interleukin (IL)-1ß in parenchymal cells. BBB damage was assessed by extravasation of intravascular fluorescein isothiocyanate-albumin. The effects of arterially perfused anakinra, a human recombinant IL-1ß receptor antagonist, were investigated on epileptiform discharges, brain inflammation, and BBB damage. RESULTS: Seizure induction in the absence of extracerebral factors promoted the release of IL-1ß from brain resident cells and enhanced its biosynthesis in astrocytes. Anakinra rapidly terminated seizures, prevented their recurrence, and resolved seizure-associated BBB breakdown. INTERPRETATION: Seizures initiate brain inflammation in glia and promote BBB damage that is independent of either leukocytes or blood-borne inflammatory molecules. Brain inflammation contributes to the duration and recurrence of seizures. This study supports the use of specific anti-inflammatory drugs in clinical conditions that present with intractable recurrent seizures.

Long-lasting pro-ictogenic effects induced in vivo by rat brain exposure to serum albumin in the absence of concomitant pathology.

PURPOSE: Dysfunction of the blood-brain barrier (BBB) is a common finding during seizures or following epileptogenic brain injuries, and experimentally induced BBB opening promotes seizures both in naive and epileptic animals. Brain albumin extravasation was reported to promote hyperexcitability by inducing astrocytes dysfunction. To provide in vivo evidence for a direct role of extravasated serum albumin in seizures independently on the pathologic context, we did the following: (1) quantified the amount of serum albumin extravasated in the rat brain parenchyma during status epilepticus (SE); (2) reproduced a similar concentration in the hippocampus by intracerebroventricular (i.c.v.) albumin injection in naive rats; (3) measured electroencephalography (EEG) activity in these rats, their susceptibility to kainic acid (KA)-induced seizures, and their hippocampal afterdischarge threshold (ADT). METHODS: Brain albumin concentration was measured in the rat hippocampus and other forebrain regions 2 and 24 h after SE by western blot analysis. Brain distribution of serum albumin or fluorescein isothiocyanate (FITC)-albumin was studied by immunohistochemistry and immunofluorescence, respectively. Naive rats were injected with rat albumin or FITC-albumin, i.c.v., to mimic the brain concentration attained after SE, or with dextran used as control. Inflammation was evaluated by immunohistochemistry by measuring glial induction of interleukin (IL)-1ß. Western blot analysis was used to measure inward rectifying potassium channel subunit Kir4.1 protein levels in the hippocampus. Seizures were induced in rats by intrahippocampal injection of 80 ng KA and quantified by EEG analysis, 2 or 24 h after rat albumin or dextran administration. ADT was measured by electrical stimulation of the hippocampus 3 months after albumin injection. In these rats, EEG was continuously monitored for 2 weeks to search for spontaneous seizures. KEY FINDINGS: The hippocampal serum albumin concentration 24 h post-SE was 0.76 ± 0.21 µm. Similar concentrations were measured in other forebrain regions, whereas no changes were found in cerebellum. The hippocampal albumin concentration was similarly reproduced in naive rats by i.c.v. administration of 500 µg/4 µl rat albumin: albumin was predominantly detected extracellularly 2 h after injection, whereas at 24 h it was visible inside pyramidal neurons and in only a few scattered chondroitin sulphate proteoglycan (NG2)-positive cells, but not in glial fibrillary acidic protein (GFAP)-positive astrocytes or CR-3 complement receptor (OX-42)-positive microglia. The presence of albumin in naive rat hippocampus was associated with induced IL-1ß in GFAP-positive astrocytes and a concomitant tissue down-regulation of Kir4.1. Spiking activity was evoked by albumin in the hippocampus lasting for 2 h. When KA was intrahippocampally applied either 2 or 24 h after albumin injection, the number of total interictal spikes in 3 h EEG recording was significantly increased by twofold on average. Three months after albumin injection, neither albumin nor inflammation was detected in brain tissue; at this time, the ADT was reduced by 50% but no spontaneous seizures were observed. SIGNIFICANCE: Transient hippocampal exposure to albumin levels similar to those attained after prominent BBB breakdown resulted in increased seizure susceptibility and long-term reduction in seizure threshold, but it did not evoke spontaneous seizures. These effects may be mediated by albumin-induced astrocytes dysfunction and the associated induction of proinflammatory molecules.

The role of the meningeal lymphatic system in local meningeal inflammation and trigeminal nociception.

A system of lymphatic vessels has been recently characterized in the meninges, with a postulated role in 'cleaning' the brain via cerebral fluid drainage. As meninges are the origin site of migraine pain, we hypothesized that malfunctioning of the lymphatic system should affect the local trigeminal nociception. To test this hypothesis, we studied nociceptive and inflammatory mechanisms in the hemiskull preparations (containing the meninges) of K14-VEGFR3-Ig (K14) mice lacking the meningeal lymphatic system. We recorded the spiking activity of meningeal afferents and estimated the local mast cells population, calcitonin gene-related peptide (CGRP) and cytokine levels as well as the dural trigeminal innervation in freshly-isolated hemiskull preparations from K14-VEGFR3-Ig (K14) or wild type C57BL/6 mice (WT). Spiking activity data have been confirmed in an acquired model of meningeal lymphatic dysfunction (AAV-mVEGFR3(1-4)Ig induced lymphatic ablation). We found that levels of the pro-inflammatory cytokine IL12-p70 and CGRP, implicated in migraine, were reduced in the meninges of K14 mice, while the levels of the mast cell activator MCP-1 were increased. The other migraine-related pro-inflammatory cytokines (basal and stimulated), did not differ between the two genotypes. The patterns of trigeminal innervation in meninges remained unchanged and we did not observe alterations in basal or ATP-induced nociceptive firing in the meningeal afferents associated with meningeal lymphatic dysfunction. In summary, the lack of meningeal lymphatic system is associated with a new balance between pro- and anti-migraine mediators but does not directly trigger meningeal nociceptive state.

Significance of developmental meningeal lymphatic dysfunction in experimental post-traumatic injury.

Understanding the pathological mechanisms unfolding after chronic traumatic brain injury (TBI) could reveal new therapeutic entry points. During the post-TBI sequel, the involvement of cerebrospinal fluid drainage through the meningeal lymphatic vessels was proposed. Here, we used K14-VEGFR3-Ig transgenic mice to analyze whether a developmental dysfunction of meningeal lymphatic vessels modifies post-TBI pathology. To this end, a moderate TBI was delivered by controlled cortical injury over the temporal lobe in male transgenic mice or their littermate controls. We performed MRI and a battery of behavioral tests over time to define the post-TBI trajectories. In vivo analyses were integrated by ex-vivo quantitative and morphometric examinations of the cortical lesion and glial cells. In post-TBI K14-VEGFR3-Ig mice, the recovery from motor deficits was protracted compared to littermates. This outcome is coherent with the observed slower hematoma clearance in transgenic mice during the first two weeks post-TBI. No other genotype-related behavioral differences were observed, and the volume of cortical lesions imaged by MRI in vivo, and confirmed by histology ex-vivo, were comparable in both groups. However, at the cellular level, post-TBI K14-VEGFR3-Ig mice exhibited an increased percentage of activated Iba1 microglia in the hippocampus and auditory cortex, areas that are proximal to the lesion. Although not impacting or modifying the structural brain damage and post-TBI behavior, a pre-existing dysfunction of meningeal lymphatic vessels is associated with morphological microglial activation over time, possibly representing a sub-clinical pathological imprint or a vulnerability factor. Our findings suggest that pre-existing mLV deficits could represent a possible risk factor for the overall outcome of TBI pathology.

Peripheral Routes to Neurodegeneration: Passing Through the Blood-Brain Barrier.

A bidirectional crosstalk between peripheral players of immunity and the central nervous system (CNS) exists. Hence, blood-brain barrier (BBB) breakdown is emerging as a participant mechanism of dysregulated peripheral-CNS interplay, promoting diseases. Here, we examine the implication of BBB damage in neurodegeneration, linking it to peripheral brain-directed autoantibodies and gut-brain axis mechanisms. As BBB breakdown is a factor contributing to, or even anticipating, neuronal dysfunction(s), we here identify contemporary pharmacological strategies that could be exploited to repair the BBB in disease conditions. Developing neurovascular, add on, therapeutic strategies may lead to a more efficacious pre-clinical to clinical transition with the goal of curbing the progression of neurodegeneration.

Developmental Dysfunction of the Central Nervous System Lymphatics Modulates the Adaptive Neuro-Immune Response in the Perilesional Cortex in a Mouse Model of Traumatic Brain Injury.

Rationale: The recently discovered meningeal lymphatic vessels (mLVs) have been proposed to be the missing link between the immune and the central nervous system. The role of mLVs in modulating the neuro-immune response following a traumatic brain injury (TBI), however, has not been analyzed. Parenchymal T lymphocyte infiltration has been previously reported as part of secondary events after TBI, suggestive of an adaptive neuro-immune response. The phenotype of these cells has remained mostly uncharacterized. In this study, we identified subpopulations of T cells infiltrating the perilesional areas 30 days post-injury (an early-chronic time point). Furthermore, we analyzed how the lack of mLVs affects the magnitude and the type of T cell response in the brain after TBI. Methods: TBI was induced in K14-VEGFR3-Ig transgenic (TG) mice or in their littermate controls (WT; wild type), applying a controlled cortical impact (CCI). One month after TBI, T cells were isolated from cortical areas ipsilateral or contralateral to the trauma and from the spleen, then characterized by flow cytometry. Lesion size in each animal was evaluated by MRI. Results: In both WT and TG-CCI mice, we found a prominent T cell infiltration in the brain confined to the perilesional cortex and hippocampus. The majority of infiltrating T cells were cytotoxic CD8+ expressing a CD44hiCD69+ phenotype, suggesting that these are effector resident memory T cells. K14-VEGFR3-Ig mice showed a significant reduction of infiltrating CD4+ T lymphocytes, suggesting that mLVs could be involved in establishing a proper neuro-immune response. Extension of the lesion (measured as lesion volume from MRI) did not differ between the genotypes. Finally, TBI did not relate to alterations in peripheral circulating T cells, as assessed one month after injury. Conclusions: Our results are consistent with the hypothesis that mLVs are involved in the neuro-immune response after TBI. We also defined the resident memory CD8+ T cells as one of the main population activated within the brain after a traumatic injury.

Modeling a Nociceptive Neuro-Immune Synapse Activated by ATP and 5-HT in Meninges: Novel Clues on Transduction of Chemical Signals Into Persistent or Rhythmic Neuronal Firing.

Extracellular ATP and serotonin (5-HT) are powerful triggers of nociceptive firing in the meninges, a process supporting headache and whose cellular mechanisms are incompletely understood. The current study aimed to develop, with the neurosimulator NEURON, a novel approach to explore in silico the molecular determinants of the long-lasting, pulsatile nature of migraine attacks. The present model included ATP and 5-HT release, ATP diffusion and hydrolysis, 5-HT uptake, differential activation of ATP P2X or 5-HT3 receptors, and receptor subtype-specific desensitization. The model also tested the role of branched meningeal fibers with multiple release sites. Spike generation and propagation were simulated using variable contribution by potassium and sodium channels in a multi-compartment fiber environment. Multiple factors appeared important to ensure prolonged nociceptive firing potentially relevant to long-lasting pain. Crucial roles were observed in: (i) co-expression of ATP P2X2 and P2X3 receptor subunits; (ii) intrinsic activation/inactivation properties of sodium Nav1.8 channels; and (iii) temporal and spatial distribution of ATP/5-HT release sites along the branches of trigeminal nerve fibers. Based on these factors we could obtain either persistent activation of nociceptive firing or its periodic bursting mimicking the pulsating nature of pain. In summary, our model proposes a novel tool for the exploration of peripheral nociception to test the contribution of clinically relevant factors to headache including migraine pain.

Ongoing Electroencephalographic Rhythms Related to Exploratory Movements in Transgenic TASTPM Mice.

BACKGROUND: The European PharmaCog study (http://www.pharmacog.org) has reported a reduction in delta (1-6 Hz) electroencephalographic (EEG) power (density) during cage exploration (active condition) compared with quiet wakefulness (passive condition) in PDAPP mice (hAPP Indiana V717F mutation) modeling Alzheimer's disease (AD) amyloidosis and cognitive deficits. OBJECTIVE: Here, we tested the reproducibility of that evidence in TASTPM mice (double mutation in APP KM670/671NL and PSEN1 M146V), which develop brain amyloidosis and cognitive deficits over aging. The reliability of that evidence was examined in four research centers of the PharmaCog study. METHODS: Ongoing EEG rhythms were recorded from a frontoparietal bipolar channel in 29 TASTPM and 58 matched "wild type" C57 mice (range of age: 12-24 months). Normalized EEG power was calculated. Frequency and amplitude of individual delta and theta frequency (IDF and ITF) peaks were considered during the passive and active conditions. RESULTS: Compared with the "wild type" group, the TASTPM group showed a significantly lower reduction in IDF power during the active over the passive condition (p < 0.05). This effect was observed in 3 out of 4 EEG recording units. CONCLUSION: TASTPM mice were characterized by "poor reactivity" of delta EEG rhythms during the cage exploration in line with previous evidence in PDAPP mice. The reliability of that result across the centers was moderate, thus unveiling pros and cons of multicenter preclinical EEG trials in TASTPM mice useful for planning future studies.

Age-dependent vascular changes induced by status epilepticus in rat forebrain: implications for epileptogenesis.

Brain inflammation, angiogenesis and increased blood-brain barrier (BBB) permeability occur in adult rodent and human epileptogenic brain tissue. We addressed the role of these events in epileptogenesis using a developmental approach since the propensity to develop spontaneous seizures, therefore the induction of epileptogenesis, is age-dependent and increases with brain maturation. Inflammation, angiogenesis and BBB permeability were studied in postnatal day (PN)9 and PN21 rats, 1 week and 4 months after pilocarpine-induced status epilepticus. Brain inflammation was evaluated by interleukin(IL)-1beta immunohistochemistry; angiogenesis was quantified by measuring the density of microvessels identified by an anti-laminin antibody or by the intraluminal signal of FITC-albumin; BBB integrity was assessed by extravascular IgG immunostaining or detection of parenchymal extravasation of FITC-albumin. Neither inflammation nor angiogenesis or changes in BBB permeability were detected in PN9 rats after status epilepticus, and these rats did not develop spontaneous seizures in adulthood as assessed by video-EEG monitoring. Differently, status epilepticus in PN21 rats induced chronic inflammation, angiogenesis and BBB leakage in the hippocampus in 62% of rats, while in the remaining rats only transient inflammation in forebrain was observed. Epilepsy developed in about 62% of PN21 rats exposed to SE and these epileptic rats showed the three phenomena concomitantly in the hippocampus. PN21 rats that did not develop epilepsy 4 months after status epilepticus, as assessed by video-EEG monitoring, they did not show inflammation, angiogenesis or BBB damage in forebrain at this time. Our data show that age-dependent vascular changes and brain inflammation induced by status epilepticus are associated with epileptogenesis, suggesting that these phenomena are implicated in the mechanisms underlying the occurrence of spontaneous seizures.

Neuropeptide Y gene therapy decreases chronic spontaneous seizures in a rat model of temporal lobe epilepsy.

Temporal lobe epilepsy remains amongst the most common and drug refractory of neurological disorders. Gene therapy may provide a realistic therapeutic approach alternative to surgery for intractable focal epilepsies. To test this hypothesis, we applied here a gene therapy approach, using a recombinant adeno-associated viral (rAAV) vector expressing the human neuropeptide Y (NPY) gene, to a progressive and spontaneous seizure model of temporal lobe epilepsy induced by electrical stimulation of the temporal pole of the hippocampus, which replicates many features of the human condition. rAAV-NPY or a control vector lacking the expression cassette (rAAV-Empty) was delivered into the epileptic rat hippocampi at an early progressive stage of the disease. Chronic epileptic rats were video-EEG monitored to establish pre-injection baseline recordings of spontaneous seizures and the effect of rAAV-NPY versus rAAV-Empty vector injection. Both non-injected stimulated controls and rAAV-empty injected rats showed a similar progressive increase of spontaneous seizure frequency consistent with epileptogenesis. The delivery of rAAV-NPY in epileptic rat brain leads to a remarkable decrease in the progression of seizures as compared to both control groups and this effect was correlated with the NPY over-expression in the hippocampus. Moreover, spontaneous seizure frequency was significantly reduced in 40% of treated animals as compared to their pre-injection baseline. Our data show that this gene therapy strategy decreases spontaneous seizures and suppresses their progression in chronic epileptic rats, thus representing a promising new therapeutic strategy.

Central nervous system lymphatic unit, immunity, and epilepsy: Is there a link?

The recent definition of a network of lymphatic vessels in the meninges surrounding the brain and the spinal cord has advanced our knowledge on the functional anatomy of fluid movement within the central nervous system (CNS). Meningeal lymphatic vessels along dural sinuses and main nerves contribute to cerebrospinal fluid (CSF) drainage, integrating the cerebrovascular and periventricular routes, and forming a circuit that we here define as the CNS-lymphatic unit. The latter unit is important for parenchymal waste clearance, brain homeostasis, and the regulation of immune or inflammatory processes within the brain. Disruption of fluid drain mechanisms may promote or sustain CNS disease, conceivably applicable to epilepsy where extracellular accumulation of macromolecules and metabolic by-products occur in the interstitial and perivascular spaces. Herein we address an emerging concept and propose a theoretical framework on: (a) how a defect of brain clearance of macromolecules could favor neuronal hyperexcitability and seizures, and (b) whether meningeal lymphatic vessel dysfunction contributes to the neuroimmune cross-talk in epileptic pathophysiology. We propose possible molecular interventions targeting meningeal lymphatic dysfunctions, a potential target for immune-mediated epilepsy.

MIM-Deficient Mice Exhibit Anatomical Changes in Dendritic Spines, Cortex Volume and Brain Ventricles, and Functional Changes in Motor Coordination and Learning.

In this study, we performed a comprehensive behavioral and anatomical analysis of the Missing in Metastasis (Mtss1/MIM) knockout (KO) mouse brain. We also analyzed the expression of MIM in different brain regions at different ages. MIM is an I-BAR containing membrane curving protein, shown to be involved in dendritic spine initiation and dendritic branching in Purkinje cells in the cerebellum. Behavioral analysis of MIM KO mice revealed defects in both learning and reverse-learning, alterations in anxiety levels and reduced dominant behavior, and confirmed the previously described deficiency in motor coordination and pre-pulse inhibition. Anatomically, we observed enlarged brain ventricles and decreased cortical volume. Although MIM expression was relatively low in hippocampus after early development, hippocampal pyramidal neurons exhibited reduced density of thin and stubby dendritic spines. Learning deficiencies can be connected to all detected anatomical changes. Both behavioral and anatomical findings are typical for schizophrenia mouse models.

Recording Electrical Brain Activity with Novel Stretchable Electrodes Based on Supersonic Cluster Beam Implantation Nanotechnology on Conformable Polymers.

BACKGROUND: Multielectrodes are implanted in central and peripheral nervous systems for rehabilitation and diagnostic purposes. The physical resistance of intracranial devices to mechanical stress is critical and fractures or electrode displacement may occur. We describe here a new recording device with stretchable properties based on Supersonic Cluster Beam Implantation (SCBI) technology with high mechanical adaptability to displacement and movement. RESULTS: The capability of SCBI-based multichannel electrodes to record brain electrical activity was compared to glass/silicon microelectrodes in acute in vitro experiments on the isolated guinea pig brain preparation. Field potentials and power frequency analysis demonstrated equal recording features for SCBI and standard electrodes. Chronic in vivo epidural implantation of the SCBI electrodes confirmed excellent long-term recording properties in comparison to standard EEG metal electrodes. Tissue biocompatibility was demonstrated by neuropathological evaluation of the brain tissue 2 months after the implantation of the devices in the subarachnoid space. CONCLUSION: We confirm the biocompatibility of novel SCBI-based stretchable electrode devices and demonstrate their suitability for recording electrical brain activity in pre-clinical settings.

Interleukin Converting Enzyme inhibition impairs kindling epileptogenesis in rats by blocking astrocytic IL-1beta production.

An enhanced production of IL-1beta in glia is a typical feature of epileptogenic tissue in experimental models and in human drug-refractory epilepsy. We show here that the selective inhibition of Interleukin Converting Enzyme (ICE), which cleaves the biologically active form of IL-1beta using VX-765, blocks kindling development in rats by preventing IL-1beta increase in forebrain astrocytes, without interfering with glia activation. The average afterdischarge duration was not altered significantly by VX-765. Up to 24 h after kindling completion and drug washout, kindled seizures could not be evoked in treated rats. VX-765 did not affect seizures or afterdischarge duration in fully kindled rats. These data indicate an antiepileptogenic effect mediated by ICE inhibition and suggest that specific anti-IL-1beta pharmacological strategies can be envisaged to interfere with epileptogenic mechanisms.

NPY gene transfer in the hippocampus attenuates synaptic plasticity and learning.

Recombinant adeno-associated viral (rAAV) vector-induced neuropeptide Y (NPY) overexpression in the hippocampus exerts powerful antiepileptic and antiepileptogenic effects in rats. Such gene therapy approach could be a valuable alternative for developing new antiepileptic treatment strategies. Future clinical progress, however, requires more detailed evaluation of possible side effects of this treatment. Until now it has been unknown whether rAAV vector-based NPY overexpression in the hippocampus alters normal synaptic transmission and plasticity, which could disturb learning and memory processing. Here we show, by electrophysiological recordings in CA1 of the hippocampal formation of rats, that hippocampal NPY gene transfer into the intact brain does not affect basal synaptic transmission, but slightly alters short-term synaptic plasticity, most likely via NPY Y2 receptor-mediated mechanisms. In addition, transgene NPY seems to be released during high frequency neuronal activity, leading to decreased glutamate release in excitatory synapses. Importantly, memory consolidation appears to be affected by the treatment. We found that long-term potentiation (LTP) in the CA1 area is partially impaired and animals have a slower rate of hippocampal-based spatial discrimination learning. These data provide the first evidence that rAAV-based gene therapy using NPY exerts relative limited effect on synaptic plasticity and learning in the hippocampus, and therefore this approach could be considered as a viable alternative for epilepsy treatment.

Ongoing Electroencephalographic Activity Associated with Cortical Arousal in Transgenic PDAPP Mice (hAPP V717F).

BACKGROUND: It has been shown that theta (6-10 Hz) and delta (1-6 Hz) ongoing electroencephalographic (EEG) rhythms revealed variations in the cortical arousal in C57 Wild Type (WT) mice during cage exploration (active condition) compared to awake quiet behavior (passive condition; IMI PharmaCog project, www.pharmacog.eu). OBJECTIVE: The objective was to test if these EEG rhythms might be abnormal in old PDAPP mice modeling Alzheimer's disease (AD) with a hAPP Indiana V717F mutation (They show abnormal neural transmission, cognitive deficits, and brain accumulation of Aß1-42). METHODS: Ongoing EEG rhythms were recorded by a frontoparietal bipolar channel in 15 PDAPP and 23 WT C57 male mice (mean age of 22.8 months ±0.4 and 0.3 standard error, respectively). EEG absolute power (density) was calculated. Frequency and amplitude of individual delta and theta frequency (IDF and ITF) peaks were considered during passive and active states in the wakefulness. RESULTS: Compared with the WT group, the PDAPP group showed higher frequency of the IDF during the passive condition and lower frequency of the ITF during the active state. Furthermore, the WT but not PDAPP group showed significant changes in the frontoparietal EEG power (IDF, ITF) during active over passive state. CONCLUSION: PDAPP mice were characterized by less changes in the brain arousal during an active state as revealed by frontoparietal EEG rhythms. Future studies will have to cross-validate the present results on large animal groups, clarify the neurophysiological underpinning of the effect, and test if the disease modifying drugs against AD amyloidosis normalize those candiate EEG biomarkers in PDAPP mice.