DR MOHAMMAD A .S KAMIL

CONSULTANT NEUROLOGIST

Is there any clinical evidence?

Experimental studies(status epilepticus,febrile seizures).

Pharmacological studies

Mechanistic insights into the action of inflammatory mediators.

Concluding remarks.

Evidences of inflammation

(1) Various markers of inflammation have been measured in epileptogenic tissue from surgically treated pharmacoresistant patients, namely in malformations of cortical development (ganglioglioma, neuroepithelial tumors, focal cortical dysplasia, tuberous sclerosis) in temporal lobe epilepsy with hippocampal sclerosis and in Rasmussen’s encephalitis.

2-Levels of various cytokines have been shown to increase transiently in the blood and cerebrospinal fluid (CSF) of patients with epilepsy after different types of seizure . Moreover, the cytokine concentration in CSF was higher than in blood,suggesting a brain origin.

(3) Anti-inflammatory treatments (adrenocorticotropic hormone (ACTH), steroids, intravenous immunoglobulin (IVIg), plasmapheresis, immunosuppressants, etc.) display anticonvulsant effects in epileptic syndromes (RE, West, Lennox–Gastau, and Landau–Kleffner syndromes) with seizures resistant to classical (AEDs).

EXPERIMENTAL STUDIES (1) The causes of brain inflammation with a special

focus on the involvement of seizures, cell death,or their association as triggering factors.

(2) The possibility that a proinflammatory state in the brain can precipitate or predispose to seizures.

(3) Whether pharmacological or genetic interference with specific inflammatory pathways in the brain can modify seizure frequency or duration, neuronal survival, and epileptogenesis.

To address these points three main models have been

used: status epilepticus

febrile seizures

mimicking systemic infection

Status epilepticus kainic acid transcriptional activation of several

proinflammatory cytokine genes, suggesting that seizure activity per se could be a trigger of brain inflammation.

pilocarpine-induced seizures in mice provoke a significant upregulation in brain parenchyma of a large variety of transcripts of inflammatory mediators including the transcriptional factor NFkB, proinflammatory cytokines, chemokines and cyclo-oxygenase (COX)-2.

electrically provoked status epilepticus induces transcription of inflammatory genes very rapidly, within 30 minutes from its onset .

This transcriptional upregulation was maximal between 6 and 12 hours, then declined to baseline expression, except for interleukin (IL)-1 .

Thus, IL-1 mRNA remained increased for up to 60 days after acute status epilepticus, a time at which rats develop spontaneous recurrent seizures.

Immunohistochemical investigations were instrumental in showing the source of brain inflammation triggered by seizures.

This study showed that both microglia and astrocytes were activated and showed raised levels of IL-1 during status epilepticus and in the chronic phase of spontaneous seizures; chronic inflammation in the epileptogenesis phase (before the onset of epileptiform activity leading to the occurrence of spontaneous seizures) was sustained mainly by astrocytes.

Two further noteworthy observations emerged from this study: (1) the involvement of endothelial cells in the production of IL-1 in epileptogenic tissue; and (2) the occurrence of inflammation in brain regions with compromised blood–brain barrier (BBB) permeability. BBB leakage was assessed by serum albumin extravasation in brain parenchyma after status epilepticus and this feature was common to human epileptogenic tissue from patients with TLE.

Thus, IL-1 and other inflammatory mediators can alter the permeability properties of the BBB ; BBB leakage, by provoking the brain entry of albumin and IgG, can trigger immune and inflammatory processes in the surrounding tissue.

Microarray studies in rat models of TLE have highlighted the inflammatory response genes as those most significantly upregulated by status epilepticus, and during the epileptogenesis phase, suggesting that these inflammatory mediators may contribute to the progression of the disease and the occurrence of spontaneous seizures.

Among these inflammatory mediators, complement factors and COX-2 have been studied in more detail.

In summary, the expression studies of inflammatory markers in experimental models and human epileptic tissue have shown most pronounced upregulation in brain parenchymal cells, in the areas of seizure origin and their generalization; seizures induce inflammation involving glia and neurons, and astrocytes chiefly contribute to sustain chronic inflammation during epileptogenesis; inflammation is associated with areas of BBB damage and neuropathology.

The evidence that inflammation precedes the development of epilepsy in experimental models of TLE suggests the possibility that it takes part in the mechanisms leading to the onset of spontaneous seizures.

Febrile seizures Prolonged and repetitive febrile seizures are closely linked to the development of mesial

TLE. Several proinflammatory cytokines, including IL-1 ,act as pyrogens after central or

systemic administration. Recent evidence points to a prominent role for this cytokine in the onset of febrile

seizures Thus, transgenic immature mice lacking IL-1R type 1 have an increased threshold of

seizure induction when exposed to hyperthermia, whereas a reduction in seizure threshold is provoked by intracerebral administration of IL-1 .

In another study, fever was induced by systemic lipopolysaccharide (LPS) administration in PN14 rats; at the peak of fever, the rats showed enhanced susceptibility to kainate seizures that was associated with increases in hippocampal levels of IL-1B. Intracerebral administration of IL-1 receptor antagonist (IL-1ra), the endogenous competitive antagonist of IL-1R type 1, rescued this phenotype by reversing the enhanced susceptibility to seizures at the peak of fever .

Moreover, the induction of fever provokes a lasting upregulation of IL- 1 in the brain

Pre-existing brain inflammation

and epilepsy One possible scenario is the establishment of an

inflammatory substrate in the brain due to an initial precipitating event such as trauma, hypoxia/ischemia, or infection.

If an injury, even when subtle, occurring at birth or during the lifetime initiates a cascade of inflammatory events, is it conceivable that this process predisposes the brain to the onset of seizures? Experimental evidence clearly shows that a large variety of insults that cause inflammation in the brain also predispose to the occurrence of seizures .

In this regard, systemic administration of LPS, a component of Gram-negative bacteria that induces systemic and brain inflammation, reduces the threshold of seizure induction in mice and rats ,and this phenotype is rescued by the administration of anti-inflammatory drugs .

Three weeks later, extracellular recordings from hippocampal slices of these rats revealed enhanced neuronal responses to afferent stimulation and to convulsant drug applications. 6–8 weeks after postnatal LPS injection, seizure susceptibility was assessed and the adult rats showed significantly greater seizure susceptibility to various convulsants, as well as increased cytokine levels and enhanced neuronal degeneration within the hippocampus after limbic seizures.

These persistent increases in seizure susceptibility were dependent upon brain induction of tumor necrosis factor (TNF)-α.

Pharmacological studies Can one target inflammatory pathways to achieve

anticonvulsant effects?

To address this question, experimental studies have used pharmacological tools to interfere selectively with the production or biological actions of inflammatory mediators.

The results obtained highlight that the outcome, either anticonvulsant or proconvulsant, depends upon the specific inflammatory pathway that is inhibited. This concept will be exemplified with a focus on IL-1 and COX-2.

INTERLEUKIN-1 This cytokine clearly mediates proconvulsant effects upon

its release from glia during seizures, thereby representing an inflammatory molecule that contributes to ictal activity.

This conclusion stems from the following evidence: (1) intracerebral application of IL-1 exerts proconvulsant actions in different seizure models ; inhibition of IL-1 synthesis using caspase-1 inhibitors (also called interleukin-1 converting enzyme) results in powerful anticonvulsant effects in kainate-treated rats and prevents the occurrence of rapid kindling ; blockade of IL-1R type 1 using IL-1ra reduces seizures induced by kainate or bicuculline.

IL-1ra was also able to block seizures induced by pilocarpine following systemic administration ; this action is likely to be mediated by the ability of IL-1ra to inhibit pilocarpine-induced release of IL-1 by monocytes, an effect that appears to be instrumental for the convulsant activity of pilocarpine.

Increased blood IL-1 would induce BBB damage by altering endothelial cell tight junctions or promoting the upregulation of adhesion molecules and their consequent interaction with T cells , thus permitting pilocarpine to enter the brain at sufficient concentrations to induce seizures. IL-1ra, by preventing these peripheral proinflammatory effects of pilocarpine, impairs the drug’s ability to cause seizures.

It appears, therefore, that the IL-1β-activated pathway plays an important role in contributing to seizures, thus raising the possibility of using anti-IL-1β treatments, which are already in clinical use for inflammatory diseases , for seizure inhibition.

CYCLO-OXYGENASE-2 Inhibition of this enzyme with nonsteroidal antiinflammatory

drugs provides an example of how inhibition of inflammation may result in harmful rather than beneficial effects.

Dichotomous effects of COX-2 blockade on seizures have been reported which appear to depend both on the type of seizure and on the time of pharmacological intervention.

Thus, if COX-2 inhibition is achieved before the induction of status epilepticus with pilocarpine or kainate, proconvulsant effects are observed .

whereas if COX-2 inhibition is achieved following status epilepticus, by injecting the selective antagonist during the epileptogenesis phase, then neuroprotection and reduced spontaneous seizure severity are observed.

Mice overexpressing the human COX-2 in neurons show more intense seizures and increased mortality after kainate, and increased susceptibility to excitotoxic damage.

Moreover, COX-2-deficient mice, or mice treated with a COX-2 inhibitor, have a reduced susceptibility to kindling .

These dual effects of COX inhibitors likely depend on their specific actions on the basal production of the various prostaglandins (PGs) and on the different profiles of PG produced during seizures in the various experimental models and in the various phases of seizure.

In this respect, PGE2 has been shown to be proconvulsant and proneurotoxic ,whereas PGF2 has inhibitory action on seizures .

Mechanistic insights into the action of

inflammatory mediators

Recent investigations have addressed the mechanisms by which inflammatory molecules can alter neuronal excitability, uncovering novel pathways of communication between glia and neurons. Most of the available studies focused on TNF-α, IL-1 and PGs.

In particular, both IL-1 and TNF-α have been show to interact with the glutamatergic system by inhibiting glutamate reuptake by astrocytes .

Moreover, TNFα induces glutamate release from astrocytes via a mechanism involving PG production , and triggers the rapid insertion of Ca2 -permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors at neuronal membranes these changes are associated with enhanced excitability and excitotoxicity.

IL-1 has been shown to increase the phosphorylation of the NR2B subunit of the NMDA receptor, thus promoting Ca influx into neurons . In particular, IL-1 induces the production of ceramide via activation of sphingomyelinase, which in turn activates the Src family of tyrosine kinases leading the NR2B phosphorylation .

The activation of this pathway underlies the proconvulsant effect of IL-1β .

Interactions of these cytokines with inhibitory neurotransmission have also been reported, as TNF-α has been shown to reduce the membrane expression of -aminobutyric acid (GABA) type A receptors , and IL-1 has been reported to inhibit GABA-mediated Cl influx into neurons .

PGs also appear to have significant effects of neuronal excitability .

Somatic and dendritic membrane excitability was reduced in CA1 pyramidal neurons when endogenous PGE2 was eliminated with a selective COX-2 inhibitor.

Accordingly, the exogenous application of PGE2 produced significant increases in frequency of firing and the amplitude of excitatory postsynaptic potentials, most likely by reducing potassium currents in CA1 neurons.

COX-2-mediated PGs synthesis also leads to the production of free radicals as intermediate products, that in turn can potentiate glutamate-mediated effects .

The production of PGE2 from TNF-α-activated astrocytes can mediate astrocytic Ca-dependent glutamate release thus possibly contributing to ictal activity and excitotoxicity .

Another mechanism by which inflammation can increase neuronal excitability is the breakdown of the BBB, as cytokines have been shown to increase BBB permeability by disrupting tight junctions, activating endothelial inducible nitric oxide synthase (iNOS) and matrix Metaloproteinases.

It is known that BBB damage can promote epileptiform activity mediated by astrocytic uptake of serum albumin, which extravasates into the brain parenchyma.

The uptake of albumin impairs astrocytic function leading to altered ionic homeostasis in the extracellular space ; moreover, the neuronal uptake of IgG appears to contribute to cell damage.

Anticonvulsant Effects of Anti-inflammatory

Strategies Nonsteroidal anti-inflammatory drugs (NSAIDs) can attenuate seizures; in

particular, ibuprofen and indomethacin reduced penicillin-induced electrocorticographic and motor seizures in rats.

a similar effect was observed with paracetamol. Aspirin also protected mice from maximal electroshock (MES)- and pentylenetetrazol-induced seizures and potentiated the anticonvulsant action of diazepam and sodium valproate.

In general, conflicting data are available on the effect of COX-1 and -2 inhibitors on seizures since prostaglandins can either reduce or promote seizures and affect neuronal cell survival differently depending on their specific type and receptor subtype interactions.

Glucocorticoids (GCs) are potent inhibitors of the transcription of genes encoding most of the proinflammatory molecules, thus representing a critical endogenous negative feedback system with anti-inflammatory properties. Accordingly, a GC receptor inhibitor increased the inflammatory reactions induced by LPS in brain and enabled IL-1 and TNF α to unveil neurotoxic effects. However, prolonged elevation of GCs (corticosterone) in the high physiologic range may induce a catabolically vulnerable state in neurons and result In an exacerbation of excitotoxic damage.

This dichotomy may explain also the paradoxical proconvulsant effects of corticosterone and dexamethasone on seizures.

Interestingly, valproate inhibits the LPS-induced activation of NF-KB and the subsequent production of TNF-α and IL-6 in monocytes and glioma cells.

Carbamazepine was shown to decrease the LPS-induced production of prostaglandins and the activation of phospholipase A in rat glial cells.

This evidence suggests that part of their anticonvulsant effects may be mediated by nonconventional anti-inflammatory actions.

CONCLUDING REMARKS The hypothesis that brain inflammation is a significant

contributor to seizures and their detrimental consequences, such as neuronal cell loss and BBB damage, is strongly supported by experimental findings and corroborated by clinical observations. Recent findings suggest that chronic inflammation may also contribute to epileptogenesis, thus predisposing the brain to the development of recurrent Seizure

The relationship between inflammation and epilepsy highlights the possibility of using specific anti-inflammatory treatments as novel therapeutic approaches to control seizures and possibly to arrest, in individuals at risk, the cascade of pathological events induced by a brain injury and leading to epilepsy.

Animal studies have shown that pharmacological interference with specific inflammatory pathways can significantly reduce seizures. It is noteworthy that some of these drugs, such as caspase-1 inhibitors or IL-1ra (Anakinra), are already in clinical use for chronic inflammatory diseases.

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