Jasper's Basic Mechanisms of the Jasper's Basic Mechanisms of the Epilepsies -neuronal interactions in ictogenesis and epileptogenesis: role of inflammatory mediators

Annamaria Vezzani1 Stephan Auvin2,3 Teresa Ravizza1 Eleonora Aronica4,5 1 Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milano, Italy 2 INSERM U676, Paris, France 3 Pediatric Neurology Department, Robert Debré Univesrity Hospital, APHP, Paris, France 4 Department (Neuro) Pathology, Academisch Medisch Centrum, Amsterdam, The Netherlands 5 The Netherlands Foundation (Stichting Epilepsie Instellingen Nederland), Heemstede, The Netherlands

Abstract In the last decade, clinical and experimental evidence provided “proof-of-concept” for involvement of inflammatory processes in the in the etiopathogenesis of . The innovative view is that brain inflammation may be a common substrate contributing to seizures in drug-resistant epilepsies of different etiologies, and recurrent seizures can per se be a major cause of long-term inflammation. Neuropathology in the epileptogenic tissue can also contribute to perpetuate inflammation. Additionally, animal studies, corroborated by clinical evidence in brain specimens resected at surgery from drug resistant , has established a major role for resident microglia, and in the inflammatory sequelae. Involvement of leukocytes has also been described in some types of epilepsy. Finally, pharmacological blockade of specific inflammatory molecules and pathways can significantly reduce seizures in experimental models of seizures and epilepsy. Emerging evidence highlights the possibility of blocking chronic inflammation using specific antiinflammatory drugs to inhibit seizures, and interfering with mechanisms involved in the underlying disease.

Over the past decade, an increasing number of observations have shown the existence of rapid regulatory and reciprocal cross-talks between neurons and glia during synaptic transmission.1 In particular, neurotransmitters released from active can stimulate receptors on glial cells, resulting in internal elevation and activation of glia, which in turn can produce a neuromodulatory response by triggering gliotransmitter release.2

When a local inflammatory reaction is triggered in the brain following an injury, microglia and astrocytes become activated and start releasing a number of proinflammatory mediators, that can alter profoundly the properties of glia via autocrine actions and perturb the glia- paracrine signalling.

Understanding which soluble mediators and what molecular mechanisms are crucially involved in these interactions is instrumental in demonstrating if an imbalance in physiological glia-neuron communication may increase neuronal network excitability and reduce cell viability.2, 3 In support of this hypothesis, there is increasing evidence that proinflammatory cytokines released by glia play prominent roles in hyperexcitability leading to precipitation and recurrence, as well as in excitotoxic cell damage associated with seizures.3, 4 Moreover, recent evidence in rodent models of childhood infections and (CNS) inflammation, points to the role of specific cytokines in determining a Page 2

chronic decreased seizure threshold, and long-term behavioral deficits, which mimic co- morbities associated with epilepsy (see below).

Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies Experimental data corroborate clinical evidence of activation of specific proinflammatory pathways in human epilepsies.5–7

This chapter reports clinical observations in drug-resistant epilepsies and experimental findings in adult and immature rodent models of seizures and epileptogenesis that causally link brain inflammation to the epileptic process. We discuss the role of specific inflammatory mediators of glia-neuron communication in the etiopathogenesis of seizures.

CLINICAL FINDINGS Accumulating evidence indicates that activation of both innate and adaptive immune systems occurs in human epilepsy. The resulting inflammatory response, which chiefly involves resident brain cells such as glia and neurons, may contribute to generation of seizures, neuronal damage and cognitive impairment.6, 7

Inflammatory and Immunological Biomarkers Levels of some cytokines such as IL-6, IL-1β and IL-1receptor antagonist (ra) transiently increase in serum or plasma after various types of seizures.8, 9 There is also evidence of seizure- dependent increases in the cerebrospinal fluid (CSF) levels of these, and other, cytokines.8, 10–14 A recent study showed increased CSF levels of IL-6 and IL-1ra after tonic- clonic seizures or prolonged partial seizures, while IL-1β levels were decreased compared to control CSF.14 This observation is at variance with previous reports showing an increase in IL-1β production, thus representing one example of the great variability observed when measuring cytokines in blood or CSF of patients with epilepsy (for review see refs. 4, 7). A major limitation of studies on CSF biomarkers (including inflammatory/immunological biomarker) is the small sample size in distinct subpopulations of epilepsy patients. In addition, time of sampling, short half-life of cytokines, storage, and type of analysis may introduce additional variability in quantification. The challenge for future studies on CSF biomarkers in neurological disorders, including epilepsy, is to define the specific molecules which monitor the dynamics of pathological processes, and combine detection of these biomarkers with outcome data to improve diagnostic and prognostic accuracy.15

Numerous studies on plasma and CSF cytokines profile after febrile seizures (FS) have the same limitations noted above. Accumulating data suggest inflammation has an important role for both the development and long-term consequences of FS.16–18 The involvement of an inflammatory response in both FS development and/or recurrence has not yet led to advances in treatment of FS. Clinical trials using non-steroidal anti inflammatory drugs did not prevent FS recurrence19, and suggest that specific inflammatory pathways should be targeted as indicated by experimental studies (see later paragraph).

Genetic Studies Several studies have examined the genetic association of IL-1β, IL-1α, and IL-1ra gene polymorphisms with epilepsy (TLE) and FS susceptibility. However these studies did not have statistical power and hence provided contradictory and inconclusive results.20, 21 Thus, there is a need for collaborative collection of a large number of patients with pediatric and adult epilepsy that can provide the statistical power that can associate inflammation and genetic susceptibility to seizures and epilepsy.20

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Antiinflammatory Drugs in Epilepsy Although the role of inflammation in human epilepsy is still hypothetical, steroids and adrenocorticotropic hormone (ACTH), with antiinflammatory and immunomodulatory Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies properties, as well as high doses of immunoglobulins have been used to treat seizures in children with Rasmussen Encephalitis (RE), West syndrome, Lennox-Gastaut and Landau Kleffner syndromes.22 In addition, some antiepileptic drugs (AEDs), such as valproate and carbamazepine, display also antiinflammatory actions.23

This evidence suggests that activation of immune/inflammatory processes in epilepsy is not a mere epiphenomenon of the underlying disease but might contribute to the pathophysiology of seizures.

Infectious and Autoimmune Diseases Different common infectious or autoimmune diseases often present with seizures, or seizures develop during the course of the disease.6, 22 Neurotropic viruses, such as the herpes viruses, have been implicated in the development of seizures6, 24 and recent studies detected human herpes viruses 6B in astrocytes of patients with hippocampal sclerosis and pregressive febrile .25 An association between childhood immunizations (such as measles, mumps, and rubella, MMR vaccination) and increased risk of FS has been suggested.6, 26

Seizures arise in different autoimmune diseases affecting CNS, such as systemic lupus erythematosus, stiff man syndrome, and Hashimoto’s encephalopathy.6, 22, 27 The detection of autoantibodies against neuronal such as voltage-gated potassium channels, decarboxylase and glutamate receptors, in subpopulations of patients with epilepsy and seizure-related diseases, supports the role of immune system dysfunction in certain forms of epilepsy.28, 29 Recently, attention has focused on the syndrome of non-infectious, non- paraneoplastic, limbic encephalitis (LE) as a precipitating event in adult-onset TLE.27, 29

Seizures have been recognized to occur in a common immunologically mediated disease, such as multiple sclerosis (MS).30 The mechanisms underlying the development of seizure in MS patients are still unclear.30 A working hypothesis is that seizures may result from cortical demyelinating lesions (with or without inflammation). However, MRI evidence of cortical inflammation has been reported to be associated with epilepsy in MS.31

The prototype of inflammatory epileptic encephalopathy is represented by RE, a severe disease characterized by focal seizures and progressive deterioration of motor and cognitive functions.32 Neuropathological features of RE include cortical inflammation restricted to one brain hemisphere, with microglial and astrocytic activation, T lymphocytes infiltration and neuronal loss.32, 33 Pardo et al.32 performed a comprehensive pathological evaluation of RE tissues and demonstrated that pathology within the affected hemisphere is multifocal and progressive. Different stages of inflammation coexist in the same patient. These observations are consistent with a progressive immune/inflammatory-mediated process involving both glia and lymphocytes, and leading to neuronal damage.32, 34 The autoimmune nature of RE was suspected when autoantibodies against glutamate- subunit 3 (GLUR3) were discovered.35 However, these autoantibodies have also been detected in other epilepsy patients with severe, early onset disease and intractable seizures, and significantly associate with seizure frequency.36 Although the aetiology and pathogenesis of this severe inflammatory disease are still enigmatic, recent studies suggest as key pathogenetic mechanism, an antigen-driven MHC class-I restricted cytotoxic CD8+ T cells-mediated attack of both neurons and astrocytes.34, 37 The therapeutic options in RE are presently represented by surgery (hemispheric disconnection) to control seizures refractory to AEDs, and long-term immunotherapy against structural and functional deterioration.38, 39

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Focal Epilepsy (non-infectious, non- autoimmune) The occurrence of a complex and sustained inflammatory reaction, involving chiefly the activation of microglia and astrocytes and the related production of proinflammatory Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies molecules, has been shown in brain tissue of patients undergoing surgery for pharmacologically refractory focal epilepsy.

Hippocampal Sclerosis Hippocampal sclerosis (HS), also known as Ammon’s horn sclerosis, is the most common neuropathological finding in patients undergoing surgery for intractable TLE. The minimal criteria for the diagnosis of HS are summarized in an ILAE commission report40 and include selective neuronal cell loss and gliosis in CA1 and end folium. Astrogliosis is a major feature of HS. It can be confirmed by immunostaining for GFAP, showing dense astrogliosis in the hilar region of the dentate gyrus as well as in Cornu Ammonis (CA) subfields where prominent neuronal loss is observed (CA3, CA1). This often includes fibrillary gliosis which supports the chronicity of the process. Microglial activation has been also shown within the in HS cases.41–44 In contrast to the prominent activation of glial cells, only few cells of adaptive immunity (CD3/CD8 positive T-lymphocytes, mainly associated with microvessels) have been detected in human TLE hippocampi.42

Crespel et al.45 reported the activation of Nuclear Factor Kappa B (NFkB), a transcriptional factor activated by inflammatory molecules and responsible for transcriptional upregulation of various proinflammatory genes, in reactive astrocytes and surviving neurons in human HS specimens. A more recent histological study42 demonstrated prominent and persistent activation the IL-1β system, both in activated microglia and astrocytes as well as in neuronal cells, thus largely confirming the findings reported in chronic epileptic rats (Figure 1).46

The activation of inflammatory pathways in human TLE is supported by gene expression profile analysis44 and includes the complement pathway, which was shown to be overexpressed by both reactive astrocytes and microglia/macrophages.47 These observations suggest the existence of a reinforcing feedback between the proinflammatory cytokine system and the components of the complement cascade, which may be critical for the propagation of the inflammatory response in human TLE with HS.

A recent study demonstrated activation of the plasminogen system, as exemplified by increased expression of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in human HS.48 This may critically influence both neuronal activity and the inflammatory response. Since IL-1β, complement components and plasminogen activators have been reported to affect the permeability properties of the BBB4, we might speculate that the leaky BBB that is observed in HS49, 50, might be partly sustained by the inflammatory actions of different molecules that surround the blood vessels.

Attention has recently focused on the role in seizure precipitation and recurrence of Toll-like receptors (TLR) signalling pathways51 (see section on experimental models and ref. 51). Interestingly, prominent and persistent overexpression of TLR4 and its endogenous ligand, high mobility group box 1 (HMGB1), has been demonstrated in glia and neurons in human TLE within the hippocampus, with a cellular distribution that confirm the findings reported in chronic epileptic mice (Figure 1).51

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Figure 1. Inflammatory processes in chronic epilepsy patients: focal epilepsy 42, 51, 65, 67 Panels A–F: hippocampal sclerosis (HS). A–B: HLA-DR immunoreactivity (IR) in the dentate region of control hippocampus (A; gcl: layer) and HS (B), showing strong and diffuse microglial activation in HS. C–D: IL-1β IR in the hilar region of control hippocampus (C) and HS (D), showing in HS expression in residual neurons (arrow) and reactive glial cells (arrows and inserts a–b; insert a shows IL-1 β immunoreactivity in perivascular astrocytic endfeet). E–F: TLR4 IR in the CA1 region of control hippocampus (E) and HS (F), showing in HS expression in residual neurons (arrows) and reactive glial cells (arrow-heads); Insert a in F: co-localization of TLR4 (red) with GFAP (green) in a reactive . Insert b in F: High Mobility Group Box 1 (HMGB1; red) IR, showing both nuclear and cytoplasmic localization in astrocytes (GFAP, green) in HS. Panels G–S: focal cortical dysplasia (FCD) type IIB. G–H: HLA-DR immunoreactivity (IR) in control cortex (G) and FCD (H), showing strong and diffuse microglial activation within the dysplastic cortex; insert a in H shows activated microglial cells (arrows) surrounding a dysmorphic neuron (arrow-head); insert b in H: merged image showing HLA-DR-positive microglial cells (green) surrounding a phospho-S6 ribosomal (pS6)-positive balloon cell (red). I–L: FCD specimen with CD3 positive cells in cortex (I) and (L); arrows in I show lymphocytes surrounding a balloon cell (asterisk) and a dysmorphic neuron (arrow-head); M: CD8-positive T lymphocytes in FCD, surrounding balloon cells (asterisks). N–O: FCD showing perivascular DC-SIGN (CD209) positive cells; O: perivascular distribution of DC-SIGN (red) and CD3 (blue) positive cells. P– S: IL-1β IR. P–Q: IL-1β IR in control cortex (P) and in FCD (Q). Strong IL-1β IR was observed in large dysplastic neurons (single arrow), glial cells (single arrowheads) and in balloon cells (double arrowheads) within the dysplastic cortex (FCD; Q); insert in

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Q: co-localization of IL-1β (red) with NeuN (green) in a dysmorphic neuron. R: co-localization of IL-1β (red) with HLA-DR (HLA; green) in a microglial cell; S: co-localization of IL-1β (red) with vimentine (VIM; green) in astroglial cells. Scale bar in A: A–D, G–H, L: 160 μm; E–F, I, M, P–Q: 80 μm. N: 20 μm. O: 40 μm; Q–R: 25 μm. Jasper's Basic Mechanisms of the EpilepsiesFocal Jasper's Basic Mechanisms of the Epilepsies Malformations of Cortical Development Tuberous Sclerosis Complex (TSC) Tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem disorder resulting from a mutation in the TSC1 or TSC2 genes.52 In addition to autism and cognitive disabilities, epilepsy is the most common neurological symptom of TSC. Epilepsy is present in 70–80% of individuals with TSC (reviewed in ref. 52). The characteristic brain lesions of TSC are cortical tubers, subependymal nodules, and subependymal giant cell tumors (SEGA).53

Activation of inflammatory pathways in cortical tubers has been suggested initially by the presence of macrophages and alterations in the expression of TNF-α,NFKB and cell adhesion molecules in these lesions.54 A subsequent study demonstrated that prominent inflammatory changes (i.e. activation of the complement cascade and IL-1β signaling) observed in these lesions include both activated microglia and astrocytes, as well as the adaptive immune response.55 In addition, focal changes in BBB permeability were detected in both cortical tubers and SEGA, as demonstrated by perivascular leakage of serum albumin, with uptake into astrocytes.55 Gene expression analysis of cortical tubers showed that inflammatory genes and cell adhesion molecules are highly expressed in tubers compared with autopsy controls.48, 56 Various potential mechanisms may underlie the presence of inflammatory cells in the TSC- associated lesions and further investigation is required to ascertain the involvement of the mTOR pathway, influencing both brain parenchymal and adaptive immune responses.57, 58

A recent study suggests that astrogliosis in tubers is a dynamic process, with progression of astrocytes from “reactive” to “gliotic”.59 In addition, apoptotic cell death is also observed in cortical tubers.54–56 Thus, the prominent activation of the inflammatory cascade could also play a role in the dynamics of TSC lesions and might contribute to progressive neuronal and glial cell injury in TSC patients.

Finally, whether the presence of a prominent population of inflammatory cells may contribute to the behavioral disorders in TSC deserves further investigation. Immunological dysfunctions and microglial activation occurring early during development have been implicated in autism which is common in TSC patients (for reviews see ref. 60).

Focal cortical dysplasia (FCD) Focal cortical dysplasias (FCD) represent sporadic architectural and cytoarchitectural malformations of the cerebral cortex, which are recognized causes of chronic medically intractable epilepsy in children and young adults.61–63 According to the current histopathological classification system, FCD have been classified into Type I, characterized by cortical dyslamination, and Type II, characterized by additional cytoarchitectural abnormalities, i.e. the presence of dysmorphic neurons and balloon cells.62 Evidence of both necrotic and apoptotic cell death has been reported in pediatric FCD cases, suggesting that intractable epilepsy in childhood due to FCD could be associated with progressive cell injury.64

Activation of astrocytes and cells of the microglia/macrophage lineage has been described in FCD specimens from both pediatric and adult patients.64–66 In a cohort of FCD patients the density of activated HLA-DR-positive microglial cells (but not CD68-positive macrophages), and the level of expression of IL-1β in parenchymal glia and neurons positively correlates with the duration of epilepsy, as well as with the frequency of seizures prior to surgical resection.66, 67 The number of HLA-DR-positive cells was significantly higher in FCD type

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II vs type I despite the absence of significant differences in seizure frequency and duration.65 Activation of complement and IL-1β signaling pathways and the chemokine MCP-1 (CCl2) in activated microglia, astrocytes, dysplastic neurons and balloon cells were

Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies also more pronounced in FCD type II.65 These results indicate that activation of inflammatory processes is not simply an effect of seizure activity, suggesting a role for the underlying neuropathology. mTOR activation observed within the cellular components of type II, but not type I FCD, could also contribute to the inflammatory response.

Some degree of activation of adaptive immunity has been also observed in FCD, and the presence of CD8+T-lymphocytes was greater in FCD II specimens than in FCD type I (Figure 1).65 In the same study dendritic cells (DCs; antigen-presenting cells involved in the initiation of the adaptive immunity) were detected around intraparenchymal blood vessels only in FCD type II specimens.65

Increased levels of proinflammatory cytokines and chemokines have been also shown in pediatric FCD cases64. There is also evidence of activation of the plasminogen-65, the TLR-68 and the VEGF-mediated signalling55, which, through different mechanisms, could contribute to glial activation and associated inflammatory reactions.

Glioneuronal Tumors Glioneuronal tumors (GNTs), including ganglioglioma (GG) and dysembryoplastic neuroepithelial tumors (DNT), are well differentiated, slowly growing neuroepithelial tumors. They are rare tumors, representing approximately 1.3% of all brain tumors.69 However, GG appear to constitute the most frequent tumor entity in young patients undergoing surgery for intractable epilepsy.70

GNTs are characterized by a mixture of dysplastic neurons and glial cells and have been included in the group of developmental disorders characterized by abnormal cell proliferation.71 Abundant population of activated microglial cells and perivascular lymphocytes represent common features of these tumors.69, 72 In both GG and DNT, a significant number of microglia/macrophages are observed within the tumor and in the peritumoral regions. The density of activated microglial cells as well as the IL-1β levels in parenchymal cells, correlate with the duration of epilepsy, as well as with the frequency of seizures prior to surgical resection.67, 72 In GG, both gene expression and immunocytochemical studies72, 73 provide evidence of a prominent activation of the inflammatory response including the presence of perivascular T lymphocyte and T cell receptor signalling pathway within the tumor.73 Upregulation of IL-1β and IL-1R1, activation of the complement cascade and the Toll-like receptor pathway in glia and neurons are consistent feature in GG.73

EXPERIMENTAL MODELS Experimental models of seizures and epilepsy were instrumental in investigating the causes of brain inflammation in human epilepsy, and in demonstrating that specific inflammatory pathways are pivotally involved in seizure precipitation and recurrence.

Models of Seizures in Adult Animals Seizures and Inflammation A novel concept emerging from experimental findings is that seizure activity in adult mice or rats triggers the synthesis and release of various proinflammatory molecules in the brain. In particular, a proinflammatory response occurs during seizures provoked by chemoconvulsants or electrical stimulations, in brain areas where seizures are generated and spread.4 This response includes a rapid onset raise in proinflammatory cytokines in glia and may involve

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also neurons (Figure 2). This phenomenon is accompanied by upregulation of TLR and their endogenous ligands, such as HMGB151, 74, followed by a cascade of downstream inflammatory events which include the activation of NFkB, chemokine production,

Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies complement system induction, increased expression of adhesion molecules.4, 47, 75–77 Upregulation of receptors for proinflammatory cytokines both in glia and neurons in seizure models suggests that both autocrine and paracrine actions take place with possible different functional outcomes (for review see ref. 4).

The astrocytic endfeet impinging on brain microvasculature and the endothelial cells of the BBB also express inflammatory molecules during seizures suggesting that brain inflammation is involved in the BBB damage described in experimental and human epileptogenic tissues (for reviews see refs. 4, 7, 78). Indeed, proinflammatory cytokines have been shown to cause disassembling of the tight junctions, the production of NO and the activation of matrix methalloproteinases in endothelial cells.3, 4, 78–80

The cascade of inflammatory mediators in brain parenchyma, chemokines production and upregulation of adhesion molecules on endothelial cells may in principle recruit immunocompetent cells from the blood stream, resulting in brain extravasation of macrophages, leukocytes and lymphocytes. However, this peripheral inflammatory component is not prominent in animal models of TLE.42, 74 Differently, T and B cells but not neurotrophils, were transiently detected in mouse brain between 24–72 h from generalized seizures induced by maximal electroshock seizure (MES).81

Importantly, seizures can induce cytokines expression independently on cell death as clearly shown in non-lesional models of seizures.17, 81–83 On the contrary, inflammation caused by seizures precedes cell loss in lesional models42 and may contribute to it. The causal role of inflammation in cell loss is supported by findings in developmental models of seizures84, and by the evidence that injections of inflammatory mediators can exacerbate apoptotic and excitotoxic cell death.85, 86

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Figure 2. Inflammatory processes in the hippocampus of epileptic mice Panels a,d: Immunohistochemical evidence of gliosis: CD11b (a,b) and GFAP (c,d) staining in control (a,c) and epileptic mice (b,d). Insets report high magnification of immunopositive glia cells. In control mice, CD11b staining was observed in resting microglia cells (a), GFAP immunoreactivity was observed in stellate-shaped astrocytes with thin processes denoting their resting state (c). In epileptic mice, CD11b denotes strongly immunopositive cells with a round shape and thick processes (b); GFAP immunostaining was increased in astrocytes exhibiting hypertrophic cell bodies and processes (d). Panels e–f2: IL-1β immunostaining control (e) and epileptic mice (f–f2). In control mice, IL-1β staining was absent (e), while in epileptic mice, it was increased in GFAP-positive parenchymal astrocytes (f; co-localization in f1: IL-1β green, GFAP red), and in perivascular astrocytes (inset in f). CD11b-positive microglia did not express IL-1β (f2: IL-1β green, CD11b red). Panels g–h2: HMGB1 immunoreactivity in control (g) and epileptic mice (h–h2). In control mice HMGB1 was observed in the nuclei of pyramidal neurons, and of scattered cells in strata radiatum-lacunoso molecolare (g and its inset). Epileptic mice have an increased number of HMGB1 positive cells (h), and HMGB1 signal is also present in the cytoplasm of cells with glial morphology (inset in h). Co-localizations show that HMGB1 is present in the cytoplasm of CD11b-positive microglia-like cells (co-localization in h1: HMGB1 green, CD11b red, Hoecsht blue), and around nuclei in GFAP-positive astrocytes (co-localization in h2: HMGB1 green, GFAP red, Hoecsht blue; see also HMGB1 alone in inset).

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Panels i–j3: TLR4 signal in control (i) and epileptic mice (j–j3). TLR4 immunoreactivity is not detected in control mice (i), while in epileptic mice is present in neurons (arrows in j; co-localization in j1: TLR4 green, NeuN red) and in GFAP-positive astrocytes (arrowheads in j; co-localization in j2: TLR4 green, GFAP red). CD11b-positive microglia does not express TLR4 (co-localization in j3: TLR4 green, CD11b red). Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies Scale bar: a–j 100μm; insets in a–j, f1, f2, h1, h2, j1–j3 20μm. Role of Inflammation in Seizures The injection of proinflammatory molecules such as IL-1β82, 87, TLR agonists51, complement system components88 or specific prostaglandins76, 89 in rodent brain, results in receptor- mediated proconvulsant effects. In contrast, the intracerebral injection of specific antagonists of some of these proinflammatory molecules, or interference with related intracellular signalling pathways, mediates powerful or neuroprotective effects.51, 87, 90– 95 Transgenic mice with perturbed cytokine signalling show significant changes in seizure susceptibility or cell damage83, 96–99, thus supporting the pharmacological evidence of a modulatory role of cytokines in neuronal excitability. IL-6 and TNF-alpha have either proconvulsant or anticonvulsant effects dependending on the cytokine receptor subtype predominantly activated and/or the specific mechanism underlying epileptic activity initiation and spread (for review see ref. 4). Similarly, COX-2 inhibition produces different outcomes on seizures depending on the experimental models.76 Proictogenic or anticonvulsant effects are likely due to the type of prostaglandins produced in the various models of seizures, thus PGF2 has anticonvulsant properties92, while PGE2 is a proneurotoxic and proconvulsant prostaglandin.89, 100

IL-1β has proconvulsant activity in different models of acute seizures; accordingly, intracerebral application of IL-1ra or inhibition of IL-1β synthesis using caspase-1 inhibitors, provide anticonvulsant effects also in models of AEDs-resistant seizures.83, 90, 91, 93, 95 (for review see ref. 4) HMGB1, an endogenous ligand of Toll-like and RAGE receptors with proinflammatory properties101, upon its release from neurons following injury or hyperexcitability, contributes to precipitation and recurrence of seizures in mice.51

Role of Inflammation in Epileptogenesis Inflammatory responses induced by brain-damaging events such as neurotrauma, , infection, febrile seizures and status epilepticus are associated with acute symptomatic seizures and a high risk of epilepsy development.102, 103 In particular, immunohistochemical analysis of IL-1β and its receptor IL-1R1 and complement factors, showed that brain inflammation induced by status epilepticus persists during epileptogenesis, and is still detectable in chronic epileptic tissue characterized by spontaneous recurrent seizures. These proinflammatory changes predominantly occur in activated microglia and astrocytes although they also involve neurons and endothelial cells of the BBB.42, 47, 51 COX-2 shows a dual profile of induction since it is induced in neurons during status epilepticus while it is significantly upregulated in astrocytes in epileptogenesis and during chronic seizures.76, 104, 105 Microarray studies have shown that proinflammatory signals linked to the immune/inflammatory response are among the biological systems mostly upregulated during epileptogenesis.106–108

The evidence of lasting brain inflammation after various pro-epileptogenic injuries 7, 103, 109, together with the established contribution of specific inflammatory mediators to seizure threshold and epileptic activity, suggest that inflammation in the brain may have a role in the development of epilepsy. Pharmacological studies were therefore designed to interfere with specific proinflammatory pathways during epileptogenesis.

In this context, different COX-2 inhibitors were tested in the lithium-pilocarpine or electrical status epilepticus models starting the treatments after status epilepticus, and continuing drug administration for different time lengths. Two studies using celecoxib or parecoxib showed

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reduction in the percentage of epileptic rats, and a decrease in spontaneous seizure frequency and duration, or reduced seizure severity, respectively. Reduced cell loss and milder microglia activation were also reported.104, 110 Conversely, no effects of COX-2 inhibitors on

Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies spontaneous seizures onset and severity or neuropathology were reported when SC8236 was used111; however, in this study longer duration of status epilepticus was allowed before treatment and drug administration during epileptogenesis was shorter.

Ravizza et al.112 showed that blockade of IL-1β biosynthesis using systemic administration of a specific ICE/caspase-1 inhibitor, prevents the acquisition of stage 5 seizures in the rapid kindling model of epileptogenesis, without changing the afterdischarge duration. After drug withdrawal, electrical stimulation did not evoke generalized seizures; moreover, drug administration in fully kindled rats did not affect stage 5 . These results suggest an antiepileptogenic effect due to inhibition of IL-1β production in astrocytes. These results are supported by lack of IL-1β immunostaining in glia of treated rats compared to control kindled rats. In contrast, anticonvulsant effects of IL-1β were reported in one study showing reduction of afterdischarge and stage 5 seizures duration in fully kindled rats after icv cytokine injection.113 Delay in kindling rate was also observed in the same study after repetitive daily intracerebroventricular (icv) injections of low doses of IL-1β. This cytokine given by icv route might have caused increased levels of glucocorticoids in response to direct HPA axis activation, which in turn could be responsible for the protective effects in kindling.

The role of leukocyte-endothelial cells adhesion mechanism in epileptogenesis was studied in lithium-pilocarpine treated mice.77 The underlying hypothesis is that leukocyte adhesion to brain microvessels would impair BBB permeability functions contributing to chronic hyperexcitability. Thus, leakage of serum albumin into brain parenchyma, and its subsequent astrocytic uptake, has been shown to decrease the K+ buffering and glutamate reuptake capacity of glia leading to ionic imbalance and increased extracellular glutamate, which would favor seizures.114, 115 The administration of specific antibodies against adhesion molecules after status epilepticus for 20 days, resulted in reduction in spontaneous seizure frequency as assessed during antibodies treatment, but no changes in their onset time or duration. Decreased neuropathology and preservation of exploratory behavior were observed in treated epileptic mice. Although these data suggest the potential involvement of leukocytes in epileptogenesis, one important caveat is that pilocarpine itself stimulates leukocytes via a primary peripheral inflammatory action, and this effect provokes BBB leakage, ionic imbalance, and allows enough systemic pilocarpine to enter the brain to trigger seizures and epileptogenesis.116 It remains therefore to be proven in additional experimental models whether this mechanism is generally operative in epilepsy.

T and B cells do not appear to play a significant role in epileptogenesis. This is suggested by two studies: one study showed that Tacrolimus, an immunosuppressant drug blocking T cell activation, did not modify spontaneous seizure onset or their frequency and duration when administered after electrically induced status epilepticus for 2 weeks.117 The other evidence showed that mice lacking T and B cells develop status epilepticus and spontaneous seizures similarly to their wild-type controls.118

Models of Seizures in Immature Animals Seizures occur more frequently early in life. FS are the most frequent etiology of seizure in childhood. In humans, initial precipitating injury including FS during childhood, are risk factors for the development of epilepsy.119 Animal models have been used to understand the underlying mechanisms of seizure occurrence and epileptogenesis in immature brain, and inflammation has emerged as a possible major contributor.

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If inflammation facilitates seizure occurrence in mature brain in almost all models, it affects seizures differently in immature brain depending on the trigger of seizures.

Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies IL-1β lowers core temperature threshold that results in seizures in a postnatal (PN) day 14 mouse model of febrile convulsions, acting on IL-1R1.16 Similarly, using a subconvulsive dose of kainate in PN14 rats, Heida & Pittman120 showed that lipopolysaccharide (LPS) exerts proconvulsant effects when the animals are febrile by favoring seizure precipitation in 50% of rats. At the onset time of seizures, IL-1β was significantly increased in the hippocampus only in rats experiencing seizures after LPS and kainate. When IL-1β was given icv in LPS-treated febrile rats, the % of animal seizing after a subconvulsant dose of kainate was significantly increased and the onset time to seizures was reduced. The opposite was found after icv injection of IL-1ra. These studies show that FS may be caused by excessive amount of IL-1β in the hippocampus.

Differently, LPS at low doses which do not increase core temperature, did not change acute susceptibility to short hyperthermic seizures in both PN11 and PN16 rats.121 However these animals develop a decreased threshold to pentylentetrazol (PTZ) when adults. A similar non- febrile dose of LPS did not exacerbate lithium-pilocarpine-induced status epilepticus at PN7 and PN14. On the contrary it induced a delay in the onset time of status epilepticus. However, these rats developed increased seizure-induced hippocampal damage.122 Non-febrile doses of LPS have been reported to decrease seizure threshold in adult mice exposed to PTZ123 and to accelerate the onset of seizures in lithium-pilocarpine treated adult rats (Auvin et al., unpublished data).

It appears from these findings that relatively low doses of LPS in immature rats, which do not cause increased core temperature, do not alter acute susceptibility to seizures although they increase seizure-induced cell loss and long-term predisposition to seizures. Moreover, the effects of LPS in immature and adult animals clearly differ since non-febrile doses exacerbate seizures in adults. The mechanisms underlying these effects, and the role played by fever and brain inflammation in determining short and long term changes in seizure threshold remain to be to be elucidated. Interactions between the LPS-induced cytokines production and the activation of HPA axis with consequent production of antiinflammatory glucocorticoids, may be important determinants of the outcomes since both phenomena are developmentally regulated and may be differently affected by the experimental setting adopted to induce seizures.

Inflammation and Seizure-induced Cell Injury in Immature Brain The relationship between seizures and neuronal injury in rodents is specific to the stage of development and the model employed to precipitate seizures. The younger is the animal, the lower is the level of seizure-induced cell injury.124, 125 The key determinants of the age- dependent occurrence of seizure-induced injury are still uncompletely understood. Recent data point to the possible involvement of inflammatory mediators, as exemplified below.

When PN9 to PN21 rats are exposed to status epilepticus induced by kainate, both cytokines expression and glia activation occur starting from the second postnatal week onwards; this temporal pattern of seizure-induced inflammation closely overlaps with that of seizure-induced cell injury. Moreover, brain inflammation precedes evidence of cell loss suggesting that proinflammatory cytokines may contribute to its occurrence.84 A causal link between inflammation and cell death in immature brain is further supported by the evidence that LPS prior to lithium-pilocarpine-induced status epilepticus, increases cell injury in hippocampus at PN7 and PN14.122 This effect occurs in the absence of changes in body temperature or in the duration of the status epilepticus.122

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These studies suggest that inflammation during post-natal development may enhance cell injury following prolonged seizures, highlighting a possibile contribution to the development of epileptogenesis. Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies Long term Consequences of Inflammation on Immature Brain Systemic or CNS inflammation by itself during a critical post-natal period is able to induce long-term changes in neuronal excitability and alterations in physiological behaviors.

When LPS was given in PN7 or PN14 rats, but not before (PN1) or after (PN20), seizure susceptibility was increased when these rats become adults, as assessed using PTZ, lithium- pilocarpine and kainate. Brain inflammation induced in PN14 mice by icv injection of polyinosinic:polycytidylic acid (Poly I:C), a TLR3 agonist which mimics viral infections, was also responsible of increase seizure susceptibility in adulthood, as shown using PTZ and lithium-pilocarpine.

Deficit in contextual fear conditioning memory was reported in both experimental settings while retention of spatial memory was affected only by the LPS treatment.

The LPS study pointed out the involvement of TNF-α and activated astrocytes126 in the long term consequences of brain inflammation, while the Poly I:C study highlights the possible involvement of IL-1β and activated microglia.127 This suggests that different inflammatory mechanisms may be activated depending on the first trigger. Interestingly, in both models of inflammation, long-term changes in hippocampal levels of several glutamate receptor subunits were reported127, 128 revealing a pathophysiological relationship between brain cytokines, glutamate receptors, behavior and seizures.

Inflammation in immature brain seems to act also as a disease modifier when it is coupled to a second hit. Systemic injection of low and non-febrile dose of LPS in PN14 rats before lithium- pilocarpine did not change the acute status epilepticus severity but resulted in more severe (stage 3–4) spontaneous seizures in adulthood.121 LPS administration did not change the number of rats that became epileptic or the frequency and duration of spontaneous seizures. No significant changes in cell number in CA1 sector was observed in LPS pretreated rats, although in some animals Fluoro-Jade positive cells were detected suggesting ongoing which was instead absent in rats not pre-exposed to LPS. A more intense reactive gliosis was also found in CA1 in rats pre-treated with LPS.129

It was found that LPS enhanced rapid kindling progression in P14 rats, and increased hippocampal excitability after kindling completion. These effects were prevented by IL-1ra indicating the involvement of IL-1β in the mechanisms of hyperexcitability.129, 130 The possibility that brain inflammation contributes to epileptogenesis, is supported by the work of Marcon et al.131 By inducing status epilepticus in PN9 and PN21 rats, these authors showed long lasting brain inflammation and vascular changes, including BBB damage and angiogenesis, only in PN21 rats but not in PN9 rats. Notably, P21 rats, but not P9 rats, show the propensity to develop epilepsy after status epilepticus.

Mechanisms of Hyperexcitability Emerging evidence has shown that non-conventional intracellular signaling pathways are activated by proinflammatory mediators in the epileptogenic tissue besides the classical induction of NFkB-mediated gene transcription described during peripheral inflammation. These novel mechanisms are likely to contribute to neuronal hyperexcitability underlying seizures, and mediate at least part of the inflammation related glia-neuron interactions. For example, cytokines can modify the function of glutamate and GABA receptors by altering receptor trafficking and their subunit assembly at neuronal membranes. Cytokines can also

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modulate glutamate receptor-mediated calcium influx in neurons by promoting AMPA- GLUR2 and NMDA-NR2B receptor subunits phosphorylation via PI3K or Src kinases, respectively.132 Recently, activation of IL-1R/Toll-like receptor signalling in neurons either

Jasper's Basic Mechanisms of the Epilepsies Jasper's Basic Mechanisms of the Epilepsies by IL-1β or by HMGB1 has been shown to play a pivotal role in seizure precipitation and recurrence via rapid Src kinases catalyzed phosphorylation of NMDA-NR2B receptors.51, 87

Cytokines and prostaglandins can also directly alter voltage-gated channels function.132 In particular, somatic and dendritic membrane excitability was significantly reduced in CA1 pyramidal neurons using a selective COX-2 inhibitor, and PGE2 produced increased firing and excitatory postsynaptic potentials, most likely by reducing potassium currents in CA1 neurons.133, 134

Activation of complement system and MAC assembly in erythrocyte membrane leads to the formation of channel conductances, resulting in Ca2+ and Na+ influx and K+ efflux, with the net effect of depolarizing the membrane potential.135 If this mechanism is also operative in neurons, it may explain why MAC assembly in the hippocampus provokes seizures.

In addition, cytokines and PGs inhibit glutamate reuptake by astrocytes136, 137 and enhance its astrocytic release138, thus resulting in increased extracellular glutamate concentration. In this regard, astrocytic glutamate release appears to contribute significantly to seizure-like events.139, 140

Finally, inflammatory mediators can also increase vascular permeability and angiogenesis (see review refs. 3, 141); their overexpression in perivascular astrocytes and endothelial cell in epilepsy may affect BBB permeability, and promote excitability in surrounding neurons.4, 78

Conclusions Clinical and experimental evidence substantiate the role of brain inflammation in the etiopathogenesis of seizures. Cinical studies show that brain inflammation is a common substrate in epilepsies of different etiologies.

Experimental studies show that recurrent seizures can trigger and perpetuate brain inflammation even in the absence of cell loss or other concomitant or pre-existing neuropathology. Long term inflammation, in turn, promotes chronic hyperexcitability, is detrimental for neuronal survival, induces behavioral dysfunctions, and may contribute to maladaptive plasticity underlying epileptogenesis.

Pharmacological studies in models of seizures and epilepsy, including models of drug-resistant epilepsy, demonstrate that interfering with specific proinflammatory pathways can effectively reduce seizures.

These findings therefore envisage novel therapy for epilepsy, by targeting specific proinflammatory pathways. This approach has two advantages, i.e. the possibility of using drugs already available in the clinical practice for septic shock or autoinflammatory autoimmune diseases, and the likelihood of interfering with a mechanism involved in the pathophysiology of seizures. This approach may therefore provides a curative, rather than merely symptomatic treatment.142

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Glia-neuronal interactions in ictogenesis and epileptogenesis: role of inflammatory mediators