Seizure-mediated iron accumulation and dysregulated iron metabolism after status epilepticus and in temporal lobe epilepsy.

Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes.

microRNA-132 is overexpressed in glia in temporal lobe epilepsy and reduces the expression of pro-epileptogenic factors in human cultured astrocytes.

Temporal lobe epilepsy (TLE) is a chronic neurological disease in humans, which is refractory to pharmacological treatment in about 30% of the patients. Reactive glial cells are thought to play a major role during the development of epilepsy (epileptogenesis) via regulation of brain inflammation and remodeling of the extracellular matrix (ECM). These processes can be regulated by microRNAs (miRs), a class of small non-coding RNAs, which can control entire gene networks at a post-transcriptional level. The expression of miRs is known to change dynamically during epileptogenesis. miR-132 is one of the most commonly upregulated miRs in animal TLE models with important roles shown in neurons. However, the possible role of miR-132 in glia remains largely unknown. The aim of this study was to characterize the cell-type specific expression of miR-132 in the hippocampus of patients with TLE and during epileptogenesis in a rat TLE model. Furthermore, the potential role of miR-132 was investigated by transfection of human primary cultured astrocytes that were stimulated with the cytokines IL-1ß or TGF-ß1. We showed an increased expression of miR-132 in the human and rat epileptogenic hippocampus, particularly in glial cells. Transfection of miR-132 in human primary astrocytes reduced the expression of pro-epileptogenic COX-2, IL-1ß, TGF-ß2, CCL2, and MMP3. This suggests that miR-132, particularly in astrocytes, represents a potential therapeutic target that warrants further in vivo investigation.

Long-lasting blood-brain barrier dysfunction and neuroinflammation after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) causes 10-20% of acquired epilepsy, which typically develops within 2 years post-injury with poorly understood mechanisms. We investigated the location, severity, evolution and persistence of blood-brain barrier (BBB) dysfunction and associated neuroinflammation after TBI, and their contribution to post-traumatic seizure susceptibility. METHODS: TBI was induced with lateral fluid-percussion in adult male Sprague-Dawley rats (6 sham, 12 TBI). Permeability of the BBB was assessed using T1-weighted magnetic resonance imaging (MRI) with gadobutrol (Gd) contrast enhancement at 4 days, 2 weeks, 2 months, and 10 months post-injury and with intravenously administered fluorescein at 11 months post-TBI. Continuous (24/7) video-EEG monitoring was performed for 3 weeks at 11 months post-injury followed by the pentylenetetrazol (PTZ) seizure-susceptibility test. In the end, rats were perfused for histology to assess albumin extravasation, iron deposits, calcifications, reactive astrocytes, microglia and monocytes. To investigate the translational value of the data obtained, BBB dysfunction and neuroinflammation were investigated immunohistochemically in autopsy brain tissue from patients with TBI and PTE. RESULTS: MRI indicated persistent Gd leakage in the impacted cortex and thalamus of variable severity in all rats with TBI which correlated with fluorescein extravasation. In the impacted cortex BBB dysfunction was evident from 4 days post-injury onwards to the end of the 10-months follow-up. In the ipsilateral thalamus, leakage was evident at 2 and 10 months post-injury. The greater the BBB leakage in the perilesional cortex at 10 months after the injury, the greater the expression of the endothelial cell antigen RECA-1 (r = 0.734, p < 0.01) and the activated macrophages/monocytes/microglia marker CD68 (r = 0.699, p < 0.05) at 11 months post-injury. Seven of the 12 rats with TBI showed increased seizure susceptibility in the PTZ-test. Unlike expected, we did not find any association between increased Gd-leakage or neuroinflammation with seizure susceptibility at 11 months post-TBI. Analysis of human autopsy tissue indicated that similar to the animal model, chronic BBB dysfunction was also evident in the perilesional cortex and thalamus of patients with PTE, characterized by presence of albumin, iron deposits and calcifications as well as markers of neuroinflammation, including reactive astrocytes, microglia and monocytes. CONCLUSIONS: Rats and humans with TBI have long-lasting cortical BBB dysfunction and neuroinflammation. Focal Gd-enhancement matched with loci of neuroinflammation, particularly in the thalamus. Although BBB leakage did not associate with increased seizure susceptibility after TBI, our data suggest that for treatments aimed to mitigate BBB damage and its secondary pathologies like chronic neuroinflammation, there is a region-specific, long-lasting therapeutic time window.

Curcumin reduces development of seizurelike events in the hippocampal-entorhinal cortex slice culture model for epileptogenesis.

OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway could be antiepileptogenic in temporal lobe epilepsy (TLE), possibly via anti-inflammatory actions. We studied effects of the mTOR inhibitor rapamycin and the anti-inflammatory compound curcumin-also reported to inhibit the mTOR pathway-on epileptogenesis and inflammation in an in vitro organotypic hippocampal-entorhinal cortex slice culture model. METHODS: Brain slices containing hippocampus and entorhinal cortex were obtained from 6-day-old rat pups and maintained in culture for up to 3 weeks. Rapamycin or curcumin was added to the culture medium from day 2 in vitro onward. Electrophysiological recordings revealed epileptiformlike activity that developed over 3 weeks. RESULTS: In week 3, spontaneous seizurelike events (SLEs) could be detected using whole cell recordings from CA1 principal neurons. The percentage of recorded CA1 neurons displaying SLEs was lower in curcumin-treated slice cultures compared to vehicle-treated slices (25.8% vs 72.5%), whereas rapamycin did not reduce SLE occurrence significantly (52%). Western blot for phosphorylated-S6 (pS6) and phosphorylated S6K confirmed that rapamycin inhibited the mTOR pathway, whereas curcumin only lowered pS6 expression at one phosphorylation site. Real-time quantitative polymerase chain reaction results indicated a trend toward lower expression of inflammatory markers IL-1ß and IL-6 and transforming growth factor ß after 3 weeks of treatment with rapamycin and curcumin compared to vehicle. SIGNIFICANCE: Our results show that curcumin suppresses SLEs in the combined hippocampal-entorhinal cortex slice culture model and suggest that its antiepileptogenic effects should be further investigated in experimental models of TLE.

Increased expression of matrix metalloproteinase 3 can be attenuated by inhibition of microRNA-155 in cultured human astrocytes.

BACKGROUND: Temporal lobe epilepsy (TLE) is a chronic neurological disease, in which about 30% of patients cannot be treated adequately with anti-epileptic drugs. Brain inflammation and remodeling of the extracellular matrix (ECM) seem to play a major role in TLE. Matrix metalloproteinases (MMPs) are proteolytic enzymes largely responsible for the remodeling of the ECM. The inhibition of MMPs has been suggested as a novel therapy for epilepsy; however, available MMP inhibitors lack specificity and cause serious side effects. We studied whether MMPs could be modulated via microRNAs (miRNAs). Several miRNAs mediate inflammatory responses in the brain, which are known to control MMP expression. The aim of this study was to investigate whether an increased expression of MMPs after interleukin-1ß (IL-1ß) stimulation can be attenuated by inhibition of the inflammation-associated miR-155. METHODS: We investigated the expression of MMP2, MMP3, MMP9, and MMP14 in cultured human fetal astrocytes after stimulation with the pro-inflammatory cytokine IL-1ß. The cells were transfected with miR-155 antagomiR, and the effect on MMP3 expression was investigated using real-time quantitative PCR and Western blotting. Furthermore, we characterized MMP3 and miR-155 expression in brain tissue of TLE patients with hippocampal sclerosis (TLE-HS) and during epileptogenesis in a rat TLE model. RESULTS: Inhibition of miR-155 by the antagomiR attenuated MMP3 overexpression after IL-1ß stimulation in astrocytes. Increased expression of MMP3 and miR-155 was also evident in the hippocampus of TLE-HS patients and throughout epileptogenesis in the rat TLE model. CONCLUSIONS: Our experiments showed that MMP3 is dynamically regulated by seizures as shown by increased expression in TLE tissue and during different phases of epileptogenesis in the rat TLE model. MMP3 can be induced by the pro-inflammatory cytokine IL-1ß and is regulated by miR-155, suggesting a possible strategy to prevent epilepsy via reduction of inflammation.

Activation of the innate immune system is evident throughout epileptogenesis and is associated with blood-brain barrier dysfunction and seizure progression.

OBJECTIVE: Because brain inflammation may contribute to the pathophysiology of temporal lobe epilepsy (TLE), we investigated the expression of various inflammatory markers of the innate and adaptive immune system in the epileptogenic human and rat hippocampus in relation to seizure activity and blood-brain barrier (BBB) dysfunction. METHODS: Immunohistochemistry was performed using various immune cell markers (for microglia, monocytes, macrophages, T lymphocytes, and dendritic cells) on hippocampal sections of drug-resistant TLE patients and patients who died after status epilepticus. The expression of these markers was also studied in the electrical post-status epilepticus rat model for TLE, during the acute, latent, and chronic epileptic phase. BBB dysfunction was assessed using albumin immunohistochemistry and the BBB tracer fluorescein. RESULTS: Monocyte infiltration, microglia, and perivascular macrophage activation were persistently increased in both epileptogenic human and rat hippocampus, whereas T lymphocytes and dendritic cells were not or were scarcely detected. In addition to this, increased expression of C-C motif ligand 2 (CCL2) and osteopontin was observed. In humans, the expression of CD68 and CCL2 was related to the duration of epilepsy and type of pathology. In rats, the expression of CD68, CCL2, and the perivascular macrophage marker CD163 was related to the duration of the initial insult and to the number of spontaneous seizures. Interestingly, the number of CD163-positive perivascular macrophages was also positively correlated to BBB dysfunction in chronic epileptic rats. SIGNIFICANCE: These data suggest a proepileptogenic role for monocytes/macrophages and other cells of the innate immune response, possibly via increased BBB leakage, and indicate that T cells and dendritic cells, which are closely associated with the adaptive immune response, are only sparsely infiltrated during epileptogenesis in the electrical post-status epilepticus rat model. Future studies should reveal the relative importance of these immune cells and whether specific manipulation can modify or prevent epileptogenesis.

Standardization procedure for plasma biomarker analysis in rat models of epileptogenesis: Focus on circulating microRNAs.

The World Health Organization estimates that globally 2.4 million people are diagnosed with epilepsy each year. In nearly 30% of these cases, epilepsy cannot be properly controlled by antiepileptic drugs. More importantly, treatments to prevent or modify epileptogenesis do not exist. Therefore, novel therapies are urgently needed. In this respect, it is important to identify which patients will develop epilepsy and which individually tailored treatment is needed. However, currently, we have no tools to identify the patients at risk, and diagnosis of epileptogenesis remains as a major unmet medical need, which relates to lack of diagnostic biomarkers for epileptogenesis. As the epileptogenic process in humans is typically slow, the use of animal models is justified to speed up biomarker discovery. We aim to summarize recommendations for molecular biomarker research and propose a standardized procedure for biomarker discovery in rat models of epileptogenesis. The potential of many phylogenetically conserved circulating noncoding small RNAs, including microRNAs (miRNAs), as biomarkers has been explored in various brain diseases, including epilepsy. Recent studies show the feasibility of detecting miRNAs in blood in both experimental models and human epilepsy. However, the analysis of circulating miRNAs in rodent models is challenging, which relates both to the lack of standardized sampling protocols and to analysis of miRNAs. We will discuss the issues critical for preclinical plasma biomarker discovery, such as documentation, blood and brain tissue sampling and collection, plasma separation and storage, RNA extraction, quality control, and RNA detection. We propose a protocol for standardization of procedures for discovery of circulating miRNA biomarkers in rat models of epileptogenesis. Ultimately, we hope that the preclinical standardization will facilitate clinical biomarker discovery for epileptogenesis in man.

Increased expression of (immuno)proteasome subunits during epileptogenesis is attenuated by inhibition of the mammalian target of rapamycin pathway.

OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway reduces epileptogenesis in various epilepsy models, possibly by inhibition of inflammatory processes, which may include the proteasome system. To study the role of mTOR inhibition in the regulation of the proteasome system, we investigated (immuno)proteasome expression during epileptogenesis, as well as the effects of the mTOR inhibitor rapamycin. METHODS: The expression of constitutive (ß1, ß5) and immunoproteasome (ß1i, ß5i) subunits was investigated during epileptogenesis using immunohistochemistry in the electrical post-status epilepticus (SE) rat model for temporal lobe epilepsy (TLE). The effect of rapamycin was studied on (immuno)proteasome subunit expression in post-SE rats that were treated for 6 weeks. (Immuno)proteasome expression was validated in the brain tissue of patients who had SE or drug-resistant TLE and the effect of rapamycin was studied in primary human astrocyte cultures. RESULTS: In post-SE rats, increased (immuno)proteasome expression was detected throughout epileptogenesis in neurons and astrocytes within the hippocampus and piriform cortex and was most evident in rats that developed a progressive form of epilepsy. Rapamycin-treated post-SE rats had reduced (immuno)proteasome protein expression and a lower number of spontaneous seizures compared to vehicle-treated rats. (Immuno)proteasome expression was also increased in neurons and astrocytes within the human hippocampus after SE and in patients with drug-resistant TLE. In vitro studies using cultured human astrocytes showed that interleukin (IL)-1ß-induced (immuno)proteasome gene expression could be attenuated by rapamycin. SIGNIFICANCE: Because dysregulation of the (immuno)proteasome system is observed before the occurrence of spontaneous seizures in rats, is associated with progression of epilepsy, and can be modulated via the mTOR pathway, it may represent an interesting novel target for drug treatment in epilepsy.

Neuroinflammatory targets and treatments for epilepsy validated in experimental models.

A large body of evidence that has accumulated over the past decade strongly supports the role of inflammation in the pathophysiology of human epilepsy. Specific inflammatory molecules and pathways have been identified that influence various pathologic outcomes in different experimental models of epilepsy. Most importantly, the same inflammatory pathways have also been found in surgically resected brain tissue from patients with treatment-resistant epilepsy. New antiseizure therapies may be derived from these novel potential targets. An essential and crucial question is whether targeting these molecules and pathways may result in anti-ictogenesis, antiepileptogenesis, and/or disease-modification effects. Therefore, preclinical testing in models mimicking relevant aspects of epileptogenesis is needed to guide integrated experimental and clinical trial designs. We discuss the most recent preclinical proof-of-concept studies validating a number of therapeutic approaches against inflammatory mechanisms in animal models that could represent novel avenues for drug development in epilepsy. Finally, we suggest future directions to accelerate preclinical to clinical translation of these recent discoveries.

Effects of rapamycin and curcumin treatment on the development of epilepsy after electrically induced status epilepticus in rats.

OBJECTIVE: Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment strategies in the electrogenic post-status epilepticus (SE) rat model. METHODS: Effects of mTOR inhibitor rapamycin were tested using the following three treatment protocols: (1) "stop-treatment"-post-SE treatment (6 mg/kg/day) was discontinued after 3 weeks; rats were monitored for 5 more weeks thereafter, (2) "pretreatment"-rapamycin (3 mg/kg/day) was applied during 3 days preceding SE; and (3) "chronic phase-treatment"-5 days rapamycin treatment (3 mg/kg/day) in the chronic phase. We also tested curcumin, an alternative mTOR inhibitor with antiinflammatory and antioxidant effects, using chronic phase treatment. Seizures were continuously monitored using video-electroencephalography (EEG) recordings; mossy fiber sprouting, cell death, and inflammation were studied using immunohistochemistry. Blood was withdrawn regularly to assess rapamycin and curcumin levels with high performance liquid chromatography (HPLC). RESULTS: Stop-treatment led to a strong reduction of seizures during the 3-week treatment and a gradual reappearance of seizures during the following 5 weeks. Three days pretreatment did not prevent seizure development, whereas 5-day rapamycin treatment in the chronic phase reduced seizure frequency. Washout of rapamycin was slow and associated with a gradual reappearance of seizures. Rapamycin treatment (both 3 and 6 mg/kg) led to body growth reduction. Curcumin treatment did not reduce seizure frequency or lead to a decrease in body weight. SIGNIFICANCE: The present study indicates that rapamycin cannot prevent epilepsy in the electrical stimulation post-SE rat model but has seizure-suppressing properties as long as rapamycin blood levels are sufficiently high. Oral curcumin treatment had no effect on chronic seizures, possibly because it did not reach the brain at adequate levels.

Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats II: Potential mechanisms.

OBJECTIVE: Blood-brain barrier (BBB) leakage may play a pro-epileptogenic role after status epilepticus. In the accompanying contrast-enhanced magnetic resonance imaging (CE-MRI) study we showed that the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduced BBB leakage and seizure activity during the chronic epileptic phase. Given rapamycin's role in growth and immune response, the potential therapeutic effects of rapamycin after status epilepticus with emphasis on brain inflammation and brain vasculature were investigated. METHODS: Seven weeks after kainic acid-induced status epilepticus, rats were perfusion fixed and (immuno)histochemistry was performed using several glial and vascular markers. In addition, an in vitro model for the human BBB was used to determine the effects of rapamycin on transendothelial electrical resistance as a measure for BBB integrity. RESULTS: (Immuno)histochemistry showed that local blood vessel density, activated microglia, and astrogliosis were reduced in rapamycin-treated rats compared to vehicle-treated rats. In vitro studies showed that rapamycin could attenuate TNFα-induced endothelial barrier breakdown. SIGNIFICANCE: These data suggest that rapamycin improves BBB function during the chronic epileptic phase by a reduction of local brain inflammation and blood vessel density that can contribute to a milder form of epilepsy.

Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats I: Magnetic resonance imaging.

OBJECTIVE: The mammalian target of rapamycin (mTOR) pathway has received increasing attention as a potential antiepileptogenic target. Treatment with the mTOR inhibitor rapamycin after status epilepticus reduces the development of epilepsy in a rat model. To study whether rapamycin mediates this effect via restoration of blood-brain barrier (BBB) dysfunction, contrast-enhanced magnetic resonance imaging (CE-MRI) was used to determine BBB permeability throughout epileptogenesis. METHODS: Imaging was repeatedly performed until 6 weeks after kainic acid-induced status epilepticus in rapamycin (6 mg/kg for 6 weeks starting 4 h after SE) and vehicle-treated rats, using gadobutrol as contrast agent. Seizures were detected using video monitoring in the week following the last imaging session. RESULTS: Gadobutrol leakage was widespread and extensive in both rapamycin and vehicle-treated epileptic rats during the acute phase, with the piriform cortex and amygdala as the most affected regions. Gadobutrol leakage was higher in rapamycin-treated rats 4 and 8 days after status epilepticus compared to vehicle-treated rats. However, during the chronic epileptic phase, gadobutrol leakage was lower in rapamycin-treated epileptic rats along with a decreased seizure frequency. This was confirmed by local fluorescein staining in the brains of the same rats. Total brain volume was reduced by this rapamycin treatment regimen. SIGNIFICANCE: The initial slow recovery of BBB function in rapamycin-treated epileptic rats indicates that rapamycin does not reduce seizure activity by a gradual recovery of BBB integrity. The reduced BBB leakage during the chronic phase, however, could contribute to the decreased seizure frequency in post-status epilepticus rats treated with rapamycin. Furthermore, the data show that CE-MRI (using step-down infusion with gadobutrol) can be used as biomarker for monitoring the effect of drug therapy in rats.

Status epilepticus, blood-brain barrier disruption, inflammation, and epileptogenesis.

Over the last 15 years, attention has been focused on dysfunction of the cerebral vasculature and inflammation as important players in epileptogenic processes, with a specific emphasis on failure of the blood-brain barrier (BBB; Fig. 1) (Seiffert et al., 2004; Marchi et al., 2007; Oby and Janigro, 2006; van Vliet et al., 2014; Vezzani et al., 2011) [3-7]. Here, we discuss how the BBB is disrupted as a consequence of SE and how this BBB breakdown may be involved in epileptogenesis. This article is part of a Special Issue entitled "Status Epilepticus".

Hippocampal subregion-specific microRNA expression during epileptogenesis in experimental temporal lobe epilepsy.

Since aberrant miRNA expression has been implicated in numerous brain diseases, we studied miRNA expression and miRNA regulation of important signaling pathways during temporal lobe epileptogenesis in order to identify possible targets for epilepsy therapy. The temporal profile of miRNA expression was analyzed in three brain regions (CA1; dentate gyrus, DG; parahippocampal cortex, PHC) associated with epileptogenesis in a rat model for temporal lobe epilepsy. Tissue was obtained after electrically-induced status epilepticus (SE) at 1day (n=5), 1week (n=5) and 3-4months (n=5), and compared with control tissue (n=10) using the Exiqon microRNA arrays which contain capture probes targeting all miRNAs for rat (p<0.01, and a 1.5 fold up- or downregulation). Expression of three blood plasma miRNAs from the same group of rats was also investigated in rats in order to determine whether plasma miRNAs could serve as potential biomarkers of the epileptogenic process. Molecular pathways potentially altered by the expression of multiple miRNAs were identified using a web-based algorithm, DIANA. In CA1 and DG, more upregulated than downregulated miRNAs were present during each stage after SE. The highest numbers of upregulated miRNAs were encountered during the chronic stage in the DG. In PHC, a high number of downregulated miRNAs were detected. Key pathways involved, based upon quantitatively altered miRNA expression were: axon guidance, MAPK signaling pathway, focal adhesion, TGFß, ErbB-, Wnt- and mTOR signaling, and regulation of actin skeleton. Expression of plasma miRNAs was differentially regulated after induction of SE. This study identified several signaling pathways possibly involved in temporal lobe epileptogenesis, not previously indicated by RNA microarray studies. These include miRNAs that regulate the ErbB and Wnt pathways and focal adhesion, which may represent interesting new targets for therapeutic interventions.

Different subsets of newborn granule cells: a possible role in epileptogenesis?

Several factors, including epileptic seizures, can strongly stimulate ongoing neurogenesis in the adult hippocampus. Although adult-born granule cells generated after seizure activity have different physiological properties from their normal counterparts, they integrate into the existing, mature network of the adult hippocampal dentate gyrus. However, the exact role of the neurogenic response during epilepsy and its possible involvement in epileptogenesis have remained elusive. Here, we discuss recent studies shedding new light on the interplay between epilepsy and neurogenesis, and try to explain discrepancies in this literature by proposing seizure severity-dependent induction of two subsets of newborn cells with different properties. We hypothesise that a low seizure intensity would stimulate neurogenesis to a 'physiological plasticity' level and have few pathological consequences. In contrast, a high initial seizure intensity may induce a specific subset of altered and/or ectopically located new granule cells with different electrophysiological properties that could initiate hyperexcitatory recurrent networks that could, in turn, contribute to chronic epilepsy. This hypothesis may clarify previously contradictory data in the literature, and could thereby aid in our understanding of the role of neurogenesis in epileptogenesis, and open up promising avenues for therapeutic intervention.

Effective reduction in seizure severity and prevention of a fatty liver by a novel low ratio ketogenic diet composition in the rapid kindling rat model of epileptogenesis.

Drug-resistant epilepsy patients may benefit from non-pharmacological therapies, such as the ketogenic diet (KD). However, its high fat content poses compliance challenges and metabolic risks. To mitigate this, we developed a novel KD composition with less fat and additional nutrients (citrate, nicotinamide riboside, and omega-3 fatty acids) for ketone-independent neuroprotection. The efficacy, metabolic and neuropathological effects of the novel KD and a classic KD were compared to a control diet in the rapid kindling model of temporal lobe epilepsy. Both KD groups entered ketosis before kindling onset, with higher ketone levels in the classic KD group. Remarkably, rats on the novel KD had slower progression of behavioral seizures as compared to rats on a control diet, while this was not the case for rats on a classic KD. Both KDs reduced electrographic after-discharge duration, preserved neurons in the dorsal hippocampus, and normalized activity in open field tests. The novel KD, despite lower fat and ketone levels, demonstrated effective reduction of behavioral seizure severity while the classic KD did not, suggesting alternative mode(s) of action are involved. Additionally, the novel KD significantly mitigated liver triglyceride and plasma fatty acid levels compared to the classic KD, indicating a reduced risk of long-term liver steatosis. Our findings highlight the potential of the novel KD to enhance therapeutic efficacy and compliance in epilepsy patients.

A novel hepatocyte ketone production assay to help the selection of nutrients for the ketogenic diet treatment of epilepsy.

The classic ketogenic diet is an effective treatment option for drug-resistant epilepsy, but its high fat content challenges patient compliance. Optimizing liver ketone production guided by a method comparing substrates for their ketogenic potential may help to reduce the fat content of the diet without loss in ketosis induction. Here, we present a liver cell assay measuring the ß-hydroxybutyrate (ßHB) yield from fatty acid substrates. Even chain albumin-conjugated fatty acids comprising between 4 and 18 carbon atoms showed a sigmoidal concentration-ßHB response curve (CRC) whereas acetate and omega-3 PUFAs produced no CRC. While CRCs were not distinguished by their half-maximal effective concentration (EC50), they differed by maximum response, which related inversely to the carbon chain length and was highest for butyrate. The assay also suitably assessed the ßHB yield from fatty acid blends detecting shifts in maximum response from exchanging medium chain fatty acids for long chain fatty acids. The assay further detected a dual role for butyrate and hexanoic acid as ketogenic substrate at high concentration and ketogenic enhancer at low concentration, augmenting the ßHB yield from oleic acid and a fatty acid blend. The assay also found propionate to inhibit ketogenesis from oleic acid and a fatty acid blend at low physiological concentration. Although the in vitro assay shows promise as a tool to optimize the ketogenic yield of a fat blend, its predictive value requires human validation.

Blood plasma inflammation markers during epileptogenesis in post-status epilepticus rat model for temporal lobe epilepsy.

PURPOSE: Brain inflammation occurs during epileptogenesis and may contribute to the development and progression of temporal lobe epilepsy. Recently, several studies have indicated that seizures may also increase specific blood plasma cytokine levels in animal models as well as in human patients with epilepsy, suggesting that peripheral inflammation may serve as a biomarker for epilepsy. Moreover, studies in epilepsy animal models have shown that peripheral inflammation may play either a pathogenic or neuroprotective role. METHODS: We evaluated the inflammatory response in blood plasma after electrically induced status epilepticus (SE) in a rat model for temporal lobe epilepsy. We measured blood plasma levels of the inflammation markers interleukin 1ß (IL-1ß), interleukin 6 (IL-6), by enzyme-linked immunosorbent assays (ELISAs) and C-reactive protein (CRP) by immunoturbidimetry, at 1 day after SE (acute period), at 1 week (during the latent period), and at 2 months after SE, which is the chronic epileptic phase when spontaneous seizures occur. Plasma levels were also measured during pilocarpine-induced SE. These were compared with plasma levels after lipopolysaccharide injection, which causes sepsis. KEY FINDINGS: Although sepsis induced a huge surge in IL-1ß and IL-6 levels, we did not detect a change in IL-1ß, IL-6, or CRP plasma levels at any time point after electrically induced SE compared to control animals. SE induced by pilocarpine produced a rise in IL-6 and CRP but not IL-1ß levels. SIGNIFICANCE: These findings suggest that plasma levels of these inflammatory proteins cannot be used as biomarkers for temporal lobe epileptogenesis.

Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 ß.

OBJECTIVE: Decreased expression of inwardly rectifying potassium (Kir) channels in astrocytes and glioma cells may contribute to impaired K⁺ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1ß (IL-1ß) on Kir4.1 mRNA and protein expression. METHODS: We investigated Kir4.1 (Kcnj10) and IL-1ß mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE), using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (n = 64), comparing the expression in tumor patients with (n = 38) and without epilepsy (n = 26). RESULTS: Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1ß mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1ß. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1ß and the cytoplasmic translocation of high mobility group box 1 (HMGB1). CONCLUSIONS: Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in particular by the inflammatory cytokine IL-1ß.

Finding a better drug for epilepsy: the mTOR pathway as an antiepileptogenic target.

The mammalian target of rapamycin (mTOR) signaling pathway regulates cell growth, differentiation, proliferation, and metabolism. Loss-of-function mutations in upstream regulators of mTOR have been highly associated with dysplasias, epilepsy, and neurodevelopmental disorders. These include tuberous sclerosis, which is due to mutations in TSC1 or TSC2 genes; mutations in phosphatase and tensin homolog (PTEN) as in Cowden syndrome, polyhydramnios, megalencephaly, symptomatic epilepsy syndrome (PMSE) due to mutations in the STE20-related kinase adaptor alpha (STRADalpha); and neurofibromatosis type 1 attributed to neurofibromin 1 mutations. Inhibition of the mTOR pathway with rapamycin may prevent epilepsy and improve the underlying pathology in mouse models with disrupted mTOR signaling, due to PTEN or TSC mutations. However the timing and duration of its administration appear critical in defining the seizure and pathology-related outcomes. Rapamycin application in human cortical slices from patients with cortical dysplasias reduces the 4-aminopyridine-induced oscillations. In the multiple-hit model of infantile spasms, pulse high-dose rapamycin administration can reduce the cortical overactivation of the mTOR pathway, suppresses spasms, and has disease-modifying effects by partially improving cognitive deficits. In post-status epilepticus models of temporal lobe epilepsy, rapamycin may ameliorate the development of epilepsy-related pathology and reduce the expression of spontaneous seizures, but its effects depend on the timing and duration of administration, and possibly the model used. The observed recurrence of seizures and epilepsy-related pathology after rapamycin discontinuation suggests the need for continuous administration to maintain the benefit. However, the use of pulse administration protocols may be useful in certain age-specific epilepsy syndromes, like infantile spasms, whereas repetitive-pulse rapamycin protocols may suffice to sustain a long-term benefit in genetic disorders of the mTOR pathway. In summary, mTOR dysregulation has been implicated in several genetic and acquired forms of epileptogenesis. The use of mTOR inhibitors can reverse some of these epileptogenic processes, although their effects depend upon the timing and dose of administration as well as the model used.