The Purinergic P2X7 Receptor as a Target for Adjunctive Treatment for Drug-Refractory Epilepsy.

Epilepsy is one of the most common neurological diseases worldwide. Anti-seizure medications (ASMs) with anticonvulsants remain the mainstay of epilepsy treatment. Currently used ASMs are, however, ineffective to suppress seizures in about one third of all patients. Moreover, ASMs show no significant impact on the pathogenic mechanisms involved in epilepsy development or disease progression and may cause serious side-effects, highlighting the need for the identification of new drug targets for a more causal therapy. Compelling evidence has demonstrated a role for purinergic signalling, including the nucleotide adenosine 5'-triphosphate (ATP) during the generation of seizures and epilepsy. Consequently, drugs targeting specific ATP-gated purinergic receptors have been suggested as promising treatment options for epilepsy including the cationic P2X7 receptor (P27XR). P2X7R protein levels have been shown to be increased in the brain of experimental models of epilepsy and in the resected brain tissue of patients with epilepsy. Animal studies have provided evidence that P2X7R blocking can reduce the severity of acute seizures and the epileptic phenotype. The current review will provide a brief summary of recent key findings on P2X7R signalling during seizures and epilepsy focusing on the potential clinical use of treatments based on the P2X7R as an adjunctive therapeutic strategy for drug-refractory seizures and epilepsy.

A Systematic Review of the Predictive and Diagnostic Uses of Neuroinflammation Biomarkers for Epileptogenesis.

A central role for neuroinflammation in epileptogenesis has recently been suggested by several investigations. This systematic review explores the role of inflammatory mediators in epileptogenesis, its association with seizure severity, and its correlation with drug-resistant epilepsy (DRE). The study analysed articles published in JCR journals from 2019 to 2024. The search strategy comprised the MESH, free terms of "Neuroinflammation", and selective searches for the following single biomarkers that had previously been selected from the relevant literature: "High mobility group box 1/HMGB1", "Toll-Like-Receptor 4/TLR-4", "Interleukin-1/IL-1", "Interleukin-6/IL-6", "Transforming growth factor beta/TGF-ß", and "Tumour necrosis factor-alpha/TNF-α". These queries were all combined with the MESH terms "Epileptogenesis" and "Epilepsy". We found 243 articles related to epileptogenesis and neuroinflammation, with 356 articles from selective searches by biomarker type. After eliminating duplicates, 324 articles were evaluated, with 272 excluded and 55 evaluated by the authors. A total of 21 articles were included in the qualitative evaluation, including 18 case-control studies, 2 case series, and 1 prospective study. As conclusion, this systematic review provides acceptable support for five biomarkers, including TNF-α and some of its soluble receptors (sTNFr2), HMGB1, TLR-4, CCL2 and IL-33. Certain receptors, cytokines, and chemokines are examples of neuroinflammation-related biomarkers that may be crucial for the early diagnosis of refractory epilepsy or may be connected to the control of epileptic seizures. Their value will be better defined by future studies.

Synaptic vesicle protein 2A mitigates parthanatos via apoptosis-inducing factor in a rat model of pharmacoresistant epilepsy.

AIMS: Synaptic vesicle protein 2A (SV2A) is a unique therapeutic target for pharmacoresistant epilepsy (PRE). As seizure-induced neuronal programmed death, parthanatos was rarely reported in PRE. Apoptosis-inducing factor (AIF), which has been implicated in parthanatos, shares a common cytoprotective function with SV2A. We aimed to investigate whether parthanatos participates in PRE and is mitigated by SV2A via AIF. METHODS: An intraperitoneal injection of lithium chloride-pilocarpine was used to establish an epileptic rat model, and phenytoin and phenobarbital sodium were utilized to select PRE and pharmacosensitive rats. The expression of SV2A was manipulated via lentivirus delivery into the hippocampus. Video surveillance was used to assess epileptic ethology. Biochemical tests were employed to test hippocampal tissues following a successful SV2A infection. Molecular dynamic calculations were used to simulate the interaction between SV2A and AIF. RESULTS: Parthanatos core index, PARP1, PAR, nuclear AIF and MIF, γ-H2AX, and TUNEL staining were all increased in PRE. SV2A is bound to AIF to form a stable complex, successfully inhibiting AIF and MIF nuclear translocation and parthanatos and consequently mitigating spontaneous recurrent seizures in PRE. Moreover, parthanatos deteriorated after the SV2A reduction. SIGNIFICANCE: SV2A protected hippocampal neurons and mitigated epileptic seizures by inhibiting parthanatos via binding to AIF in PRE.

Hypometabolism in the Posteromedial Temporal and Medial Occipital Cortex on Preoperative 2-Deoxy-2-(18F) Fluoro-D-Glucose Positron Emission Tomography Suggests Exacerbation of Visual Field Defects After Surgery for Temporal Lobe Epilepsy: A Retrospective Long-Term Follow-Up Study.

OBJECTIVE: Surgery is a good treatment option for drug-resistant temporal lobe epilepsy (TLE). 2-deoxy-2-(18F) fluoro-D-glucose (FDG) positron emission tomography (PET) is used to detect epileptic foci as hypometabolic lesions in presurgical evaluation. Visual field defects (VFDs) in the contralateral homonymous upper quadrant are common postoperative complications in TLE. This study aimed to quantify VFDs using pattern deviation probability plots (PDPPs) and examine the effect of hypometabolism in FDG-PET on VFDs. METHODS: This study included 40 patients. Both visual fields were assessed using the Humphrey field analyzer preoperatively and 3 months and 2 years postoperatively. PDPPs with <0.5% confidence level counted in the contralateral homonymous upper quadrant. FDG-PET results were compared between groups with (15 patients) and without (24 patients) hypometabolism in the optic radiation. RESULTS: All 40 patients were evaluated by Humphrey field analyzer at 3 months postoperatively and 39 at 2 years postoperatively. The incidence of VFDs 3 months postoperatively was 35/40 (87.5%), and 17/40 (42.5%) patients had severe VFDs. In cases of surgery on the left temporal lobe, ipsilateral eyes appeared to be more significantly affected than contralateral eyes. VFDs were more severe in patients with FDG hypometabolism than in those without hypometabolism in posteromedial temporal and medial occipital cortex (P < 0.01); however, 85% of patients with FDG hypometabolism had a reduced VFD 2 years postoperatively. CONCLUSIONS: PDPP counting is useful for quantifying VFDs. Preoperative dysfunction indicated by preoperative FDG-PET in the posteromedial temporal and medial occipital cortex could enhance VFDs early after TLE surgery.

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.

Analysis of the action mechanisms and targets of herbal anticonvulsants highlights opportunities for therapeutic engagement with refractory epilepsy.

Epilepsy is a neurological disorder characterized by spontaneous and recurring seizures. It poses significant therapeutic challenges due to diverse etiology, pathobiology, and pharmacotherapy-resistant variants. The anticonvulsive effects of herbal leads with biocompatibility and toxicity considerations have attracted much interest, inspiring mechanistic analysis with the view of their use for engagement of new targets and combination with antiseizure pharmacotherapies. This article presents a comprehensive overview of the key molecular players and putative action mechanisms of the most common antiepileptic herbals demonstrated in tissue culture and preclinical models. From the review of the literature, it emerges that their effects are mediated via five distinct mechanisms: (1) reduction of membrane excitability through inhibition of cation channels, (2) improvement of mitochondrial functions with antioxidant effects, (3) enhancement in synaptic transmission mediated by GABAA receptors, (4) improvement of immune response with anti-inflammatory action, and (5) suppression of protein synthesis and metabolism. While some of the primary targets and action mechanisms of herbal anticonvulsants (1, 3) are shared with antiseizure pharmacotherapies, herbal leads also engage with distinct mechanisms (2, 4, and 5), suggesting new drug targets and opportunities for their integration with antiseizure medications. Addressing outstanding questions through research and in silico modeling should facilitate the future use of herbals as auxiliary therapy in epilepsy and guide the development of treatment of pharmacoresistant seizures through rigorous trials and regulatory approval.

Differential Expression of the ß3 Subunit of Voltage-Gated Ca2+ Channel in Mesial Temporal Lobe Epilepsy.

The purpose of this study was to identify and validate new putative lead drug targets in drug-resistant mesial temporal lobe epilepsy (mTLE) starting from differentially expressed genes (DEGs) previously identified in mTLE in humans by transcriptome analysis. We identified consensus DEGs among two independent mTLE transcriptome datasets and assigned them status as "lead target" if they (1) were involved in neuronal excitability, (2) were new in mTLE, and (3) were druggable. For this, we created a consensus DEG network in STRING and annotated it with information from the DISEASES database and the Target Central Resource Database (TCRD). Next, we attempted to validate lead targets using qPCR, immunohistochemistry, and Western blot on hippocampal and temporal lobe neocortical tissue from mTLE patients and non-epilepsy controls, respectively. Here we created a robust, unbiased list of 113 consensus DEGs starting from two lists of 3040 and 5523 mTLE significant DEGs, respectively, and identified five lead targets. Next, we showed that CACNB3, a voltage-gated Ca2+ channel subunit, was significantly regulated in mTLE at both mRNA and protein level. Considering the key role of Ca2+ currents in regulating neuronal excitability, this suggested a role for CACNB3 in seizure generation. This is the first time changes in CACNB3 expression have been associated with drug-resistant epilepsy in humans, and since efficient therapeutic strategies for the treatment of drug-resistant mTLE are lacking, our finding might represent a step toward designing such new treatment strategies.

The genomic landscape across 474 surgically accessible epileptogenic human brain lesions.

Understanding the exact molecular mechanisms involved in the aetiology of epileptogenic pathologies with or without tumour activity is essential for improving treatment of drug-resistant focal epilepsy. Here, we characterize the landscape of somatic genetic variants in resected brain specimens from 474 individuals with drug-resistant focal epilepsy using deep whole-exome sequencing (>350×) and whole-genome genotyping. Across the exome, we observe a greater number of somatic single-nucleotide variants in low-grade epilepsy-associated tumours (7.92 ± 5.65 single-nucleotide variants) than in brain tissue from malformations of cortical development (6.11 ± 4 single-nucleotide variants) or hippocampal sclerosis (5.1 ± 3.04 single-nucleotide variants). Tumour tissues also had the largest number of likely pathogenic variant carrying cells. low-grade epilepsy-associated tumours had the highest proportion of samples with one or more somatic copy-number variants (24.7%), followed by malformations of cortical development (5.4%) and hippocampal sclerosis (4.1%). Recurring somatic whole chromosome duplications affecting Chromosome 7 (16.8%), chromosome 5 (10.9%), and chromosome 20 (9.9%) were observed among low-grade epilepsy-associated tumours. For germline variant-associated malformations of cortical development genes such as TSC2, DEPDC5 and PTEN, germline single-nucleotide variants were frequently identified within large loss of heterozygosity regions, supporting the recently proposed 'second hit' disease mechanism in these genes. We detect somatic variants in 12 established lesional epilepsy genes and demonstrate exome-wide statistical support for three of these in the aetiology of low-grade epilepsy-associated tumours (e.g. BRAF) and malformations of cortical development (e.g. SLC35A2 and MTOR). We also identify novel significant associations for PTPN11 with low-grade epilepsy-associated tumours and NRAS Q61 mutated protein with a complex malformation of cortical development characterized by polymicrogyria and nodular heterotopia. The variants identified in NRAS are known from cancer studies to lead to hyperactivation of NRAS, which can be targeted pharmacologically. We identify large recurrent 1q21-q44 duplication including AKT3 in association with focal cortical dysplasia type 2a with hyaline astrocytic inclusions, another rare and possibly under-recognized brain lesion. The clinical-genetic analyses showed that the numbers of somatic single-nucleotide variant across the exome and the fraction of affected cells were positively correlated with the age at seizure onset and surgery in individuals with low-grade epilepsy-associated tumours. In summary, our comprehensive genetic screen sheds light on the genome-scale landscape of genetic variants in epileptic brain lesions, informs the design of gene panels for clinical diagnostic screening and guides future directions for clinical implementation of epilepsy surgery genetics.

Dysfunction of ABC Transporters at the Surface of BBB: Potential Implications in Intractable Epilepsy and Applications of Nanotechnology Enabled Drug Delivery.

Epilepsy is a chronic neurological disorder affecting 70 million people globally. One of the fascinating attributes of brain microvasculature is the (BBB), which controls a chain of distinct features that securely regulate the molecules, ions, and cells movement between the blood and the parenchyma. The barrier's integrity is of paramount importance and essential for maintaining brain homeostasis, as it offers both physical and chemical barriers to counter pathogens and xenobiotics. Dysfunction of various transporters in the (BBB), mainly ATP binding cassette (ABC), is considered to play a vital role in hampering the availability of antiepileptic drugs into the brain. ABC (ATP-binding cassette) transporters constitute a most diverse protein superfamily, which plays an essential part in various biological processes, including cell homeostasis, cell signaling, uptake of nutrients, and drug metabolism. Moreover, it plays a crucial role in neuroprotection by out-flowing various internal and external toxic substances from the interior of a cell, thus decreasing their buildup inside the cell. In humans, forty-eight ABC transporters have been acknowledged and categorized into subfamilies A to G based on their phylogenetic analysis. ABC subfamilies B, C, and G, impart a vital role at the BBB in guarding the brain against the entrance of various xenobiotic and their buildup. The illnesses of the central nervous system have received a lot of attention lately Owing to the existence of the BBB, the penetration effectiveness of most CNS medicines into the brain parenchyma is very limited (BBB). In the development of neurological therapies, BBB crossing for medication delivery to the CNS continues to be a major barrier. Nanomaterials with BBB cross ability have indeed been extensively developed for the treatment of CNS diseases due to their advantageous properties. This review will focus on multiple possible factors like inflammation, oxidative stress, uncontrolled recurrent seizures, and genetic polymorphisms that result in the deregulation of ABC transporters in epilepsy and nanotechnology-enabled delivery across BBB in epilepsy.

Effects of GABAB receptor positive allosteric modulator BHF177 and IRS-1 on apoptosis of hippocampal neurons in rats with refractory epilepsy via the PI3K/Akt pathway.

The present study was conducted to determine the effects of the γ-aminobutyric acid B (GABAB ) receptor positive allosteric modulator BHF177 on refractory epilepsy (RE). An RE rat model was initially established via treatment with lithium-pilocarpine. The RE rats were then treated with BHF177 or the GABAB receptor antagonist CGP46381, followed by recording of their seizure rate and assessment of their spatial learning in the Morris water maze test. Treatment of BHF177 reduced the seizure intensity, whereas this effect was revered upoj treatment with CGP46381. Immunohistochemistry revealed that BHF177 treatment diminished P-glycoprotein (P-gp) expression in the hippocampal tissues of RE rats. Next, we found that BHF177 activated GABAB receptor, resulting in upregulated expression of insulin receptor substrate 1 (IRS-1) and PI3K, as well as antiapoptotic factors (Bcl-2 and mTOR), along with suppression of the apoptosis factors Bax and cleaved caspase-3 in the hippocampal tissues. Further, activation of GABAB receptors by BHF177 alleviated the inflammatory response in hippocampal tissues of RE rats, as evidenced by reduced VCAM-1, ICAM-1, and tumor necrosis factor-α levels. Next, we treated primary cultured rat hippocampal neurons with BHF177 and the IRS-1 selective inhibitor NT157. BHF177 inhibited hippocampal apoptosis in rat hippocampal neurons by regulating the IRS-1/PI3K/Akt axis through crosstalk between GABAB and insulin-like growth factor-1 receptors. Collectively, our findings indicate that the BHF177 inhibited neuron apoptosis, thus protecting against RE through the IRS-1/PI3K/Akt axis, which may present a new therapeutic channel for RE.

Functionalized PEG-PLA nanoparticles for brain targeted delivery of ketoconazole contribute to pregnane X receptor overexpressing in drug-resistant epilepsy.

OBJECTIVE: To develop a functionalized PEG-PLA nanoparticle system containing ketoconazole (KCZ) to overcome the overactivity of pregnane X receptor (PXR) for the treatment of drug-resistant epilepsy (DRE). SIGNIFICANCE: KCZ was developed as a therapy strategy for DRE limited by its lethal hepatotoxicity and minute brain concentration. KCZ-incorporated nanoparticles modified with angiopep-2 (NPs/KCZ) could reduce adverse effects of KCZ and achieve epileptic foci-targeted drug delivery. METHODS: NPs/KCZ was prepared by thin-film hydration method and characterized in vitro and in vivo. The efficacy evaluation of NPs/KCZ was evaluated in a kainic acid (KA)-induced mice model of epilepsy with carbamazepine (CBZ) treatment. RESULTS: The mean particle size and Zeta potential of NPs/KCZ were 17.84 ± 0.33 nm and - 2.28 ± 0.12 mV, respectively. The drug-loading (DL%) of KCZ in nanoparticles was 8.96 ± 0.12 % and the entrapment efficiency (EE%) was 98.56 ± 0.02 %. The critical value of critical micelle concentration was 10-3.3 mg/ml. No obvious cytotoxicity was found in vitro. The behavioral and electrographic seizure activities were obviously attenuated in NPs/KCZ+CBZ group. The CBZ concentration of brain tissues in mice treated with NPs/KCZ+CBZ was significantly increased than those treated with CBZ alone (P = 0.0028). A significantly decreased expression level of PXR and its downstream proteins was observed in NPs/KCZ+CBZ group compared with that in the control and CBZ group (All P < 0.05). CONCLUSION: Our results showed that NPs/KCZ achieved the epileptic foci-targeted delivery of KCZ and ameliorated the efficacy of CBZ on DRE by attenuating the overactivity of PXR.

The Prevention and Reversal of a Phenytoin-Resistant Model by N-acetylcysteine Therapy Involves the Nrf2/P-Glycoprotein Pathway at the Blood-Brain Barrier.

The transporter hypothesis is one of the most popular hypotheses of drug-resistant epilepsy (DRE). P-glycoprotein (P-gp), a channel protein at the blood-brain barrier (BBB), plays an important role in the transport of some anti-seizure drugs from brain tissue into vessels, which reduces drug concentrations and diminishes the effects of drug treatment. We performed this study to test whether P-gp is overexpressed in DRE and identify ways to prevent and reverse DRE. In this study, we established a phenytoin (PHT)-resistant mouse model and revealed that P-gp was overexpressed at the BBB in PHT-resistant mice. The P-gp inhibitor nimodipine decreased the resistance of phenytoin. Antioxidative preventive treatment with N-acetylcysteine (NAC) prevented the mice from entering a PHT-resistant state, and NAC therapy tended to reverse PHT resistance into sensitivity. We were also able to induce PHT resistance by activating the Nrf2/P-gp pathway, which indicates that oxidative stress plays an important role in drug resistance. Taken together, these findings suggest that antioxidative therapy may be a promising strategy for overcoming DRE.

The Effect of the Ketogenic Diet on Adiponectin, Omentin and Vaspin in Children with Drug-Resistant Epilepsy.

BACKGROUND: Changes in adipokine secretion may be involved in the anti-epileptic effect of a ketogenic diet (KD) in drug-resistant epilepsy (DRE). OBJECTIVES: The assessment of the influence of KD on serum adiponectin, omentin-1, and vaspin in children with DRE. METHODS: Anthropometric measurements (weight, height, BMI, and waist-to-hip circumference ratio) were performed in 72 children aged 3-9 years, divided into 3 groups: 24 children with DRE treated with KD, 26-treated with valproic acid (VPA), and a control group of 22 children. Biochemical tests included fasting glucose, insulin, beta-hydroxybutyric acid, lipid profile, aminotransferases activities, and blood gasometry. Serum levels of adiponectin, omentin-1 and vaspin were assayed using commercially available ELISA tests. RESULTS: Serum levels of adiponectin and omentin-1 in the KD group were significantly higher and vaspin-lower in comparison to patients receiving VPA and the control group. In all examined children, serum adiponectin and omentin-1 correlated negatively with WHR and serum triglycerides, insulin, fasting glucose, and HOMA-IR. Vaspin levels correlated negatively with serum triglycerides and positively with body weight, BMI, fasting glucose, insulin, and HOMA-IR. CONCLUSION: One of the potential mechanisms of KD in children with drug-resistant epilepsy may be a modulation of metabolically beneficial and anti-inflammatory adipokine levels.

Profiling PI3K-AKT-MTOR variants in focal brain malformations reveals new insights for diagnostic care.

Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.

Transcriptomic profile of epileptic children treated with ketogenic therapies.

Background: Ketogenic dietary therapies (KDT) are used as a treatment in childhood epilepsy. However, their mechanism has not yet been established. The main objective of this study was to determine the changes in the transcriptomic profile induced by KDT in children with epilepsy in order to shed light on its possible mechanisms. Methods: Eight children with refractory epilepsy were enrolled in the study. Peripheral blood mononuclear cells were obtained before and after the children were treated with KDT for a minimum of 6 months. RNA was extracted and mRNA and miRNA profiling were performed and analyzed. Results: Our intervention with KDT significantly reduced the seizure number in seven of the eight paediatric patients treated and caused important changes in their gene expression profile. Our study reveals modifications in the transcription of 4630 genes and 230 miRNAs. We found that the genes involved in the protection against epileptic crises were among those mainly changed. These genes collectively encode for ion channels, neurotransmitter receptors, and synapse structural proteins. Conclusions: Together our results explain the possible mechanisms of KDT and reinforce its clinical importance in the treatment of epilepsy.

Th17 cell-mediated immune response in a subpopulation of dogs with idiopathic epilepsy.

BACKGROUND: Canine idiopathic epilepsy (IE) is a common neurological disease with severe impact on the owner´s and the dog's quality of life. A subpopulation of dogs with IE does not respond to antiseizure drugs (non-responder). Th17 cells (T helper cells) and their proinflammatory Interleukin-17 (IL-17) are part of the immune system and previous studies showed their involvement in the pathogenesis of several autoimmune diseases. Non-responder might have an abnormal immune response against structures of the central nervous system. To discover a new aetiology of canine IE and thereby optimising the therapy of intractable IE, this prospective study aimed to investigate Th17 cells and IL-17 in dogs with IE. The underlying hypothesis was that in some dogs with IE a Th17 cell-mediated immune response could be detectable. METHODS: 57 dogs with IE and 10 healthy dogs (control group, C) were enrolled in the study. EDTA blood was taken to measure Th17 cells by flow cytometry. IL-17 was measured in 35 cerebrospinal fluid (CSF) and 33 serum samples using an enzyme-linked immunosorbent assay (ELISA). It was investigated whether there was a significant increase of stimulated Th17 cells in blood samples or of IL-17 in serum and CSF samples of dogs with IE in comparison to C. Correlations between the amount of Th17 cells/µL or IL-17 and different clinical parameters e.g. seizure frequency, seizure type, seizure severity or treatment response were evaluated. Additionally, Th17 cells/µL were randomly controlled of 17 dogs with IE and were examined for changes over time and in relation to treatment response. RESULTS: Ten dogs with IE had strongly elevated stimulated Th17 cells/µL within the blood (>100 Th17 cells/µL). A slight positive correlation between stimulated Th17 cells/µL and seizure severity (p = 0.046; rSpear = 0.27) was proven in these dogs. In addition, 4/10 dogs with elevated Th17 levels experienced cluster seizures and status epilepticus in comparison to 9% of the dogs with non-elevated Th17 levels (<100 Th17 cells/µL). Dogs with IE had significantly higher IL-17 values in CSF and serum samples compared to C (p<0.001; p<0.002; respectively). CONCLUSION: In single dogs with IE, strongly increased amounts of Th17 cells were detectable and dogs with elevated Th17 cells seemed to have a greater risk for experiencing a combination of cluster seizures and status epilepticus. Therefore, an underlying Th17-cell mediated immune response was suspected and hence anti-inflammatory drugs could be indicated in these single cases with intractable epilepsy.

Identification of New Compounds with Anticonvulsant and Antinociceptive Properties in a Group of 3-substituted (2,5-dioxo-pyrrolidin-1-yl)(phenyl)-Acetamides.

We report herein a series of water-soluble analogues of previously described anticonvulsants and their detailed in vivo and in vitro characterization. The majority of these compounds demonstrated broad-spectrum anticonvulsant properties in animal seizure models, including the maximal electroshock (MES) test, the pentylenetetrazole-induced seizure model (scPTZ), and the psychomotor 6 Hz (32 mA) seizure model in mice. Compound 14 showed the most robust anticonvulsant activity (ED50 MES = 49.6 mg/kg, ED50 6 Hz (32 mA) = 31.3 mg/kg, ED50scPTZ = 67.4 mg/kg). Notably, it was also effective in the 6 Hz (44 mA) model of drug-resistant epilepsy (ED50 = 63.2 mg/kg). Apart from favorable anticonvulsant properties, compound 14 revealed a high efficacy against pain responses in the formalin-induced tonic pain, the capsaicin-induced neurogenic pain, as well as in the oxaliplatin-induced neuropathic pain in mice. Moreover, compound 14 showed distinct anti-inflammatory activity in the model of carrageenan-induced aseptic inflammation. The mechanism of action of compound 14 is likely complex and may result from the inhibition of peripheral and central sodium and calcium currents, as well as the TRPV1 receptor antagonism as observed in the in vitro studies. This lead compound also revealed beneficial in vitro ADME-Tox properties and an in vivo pharmacokinetic profile, making it a potential candidate for future preclinical development. Interestingly, the in vitro studies also showed a favorable induction effect of compound 14 on the viability of neuroblastoma SH-SY5Y cells.

Activated N-methyl-D-aspartate receptor ion channels detected in focal epilepsy with [18 F]GE-179 positron emission tomography.

OBJECTIVE: Imaging activated glutamate N-methyl-D-aspartate receptor ion channels (NMDAR-ICs) using positron emission tomography (PET) has proved challenging due to low brain uptake, poor affinity and selectivity, and high metabolism and dissociation rates of candidate radioligands. The radioligand [18 F]GE-179 is a known use-dependent marker of NMDAR-ICs. We studied whether interictal [18 F]GE-179 PET would detect foci of abnormal NMDAR-IC activation in patients with refractory focal epilepsy. METHODS: Ten patients with refractory focal epilepsy and 18 healthy controls had structural magnetic resonance imaging (MRI) followed by a 90-min dynamic [18 F]GE-179 PET scan with simultaneous electroencephalography (EEG). PET and EEG findings were compared with MRI and previous EEGs. Standard uptake value (SUV) images of [18 F]GE-179 were generated and global gray matter uptake was measured for each individual. To localize focal increases in uptake of [18 F]GE-179, the individual SUV images were interrogated with statistical parametric mapping in comparison to a normal database. Additionally, individual healthy control SUV images were compared with the rest of the control database to determine their prevalence of increased focal [18 F]GE-179 uptake. RESULTS: Interictal [18 F]GE-179 PET detected clusters of significantly increased binding in eight of 10 patients with focal epilepsy but none of the controls. The number of clusters of raised [18 F]GE-179 uptake in the patients with epilepsy exceeded the focal abnormalities revealed by the simultaneously recorded EEG. Patients with extensive clusters of raised [18 F]GE-179 uptake showed the most abnormal EEGs. SIGNIFICANCE: Detection of multiple foci of abnormal NMDAR-IC activation in 80% of our patients with refractory focal epilepsy using interictal [18 F]GE-179 PET could reflect enhanced neuronal excitability due to chronic seizure activity. This indicates that chronic epileptic activity is associated with abnormal NMDAR ion channel activation beyond the initial irritative zones. [18 F]GE-179 PET could be a candidate marker for identifying pathological brain areas in patients with treatment-resistant focal epilepsy.

ABCB1 c.3435C > T and EPHX1 c.416A > G polymorphisms influence plasma carbamazepine concentration, metabolism, and pharmacoresistance in epileptic patients.

The gene polymorphisms of ABCB1, EPHX1, and SCN1A were found to influence carbamazepine (CBZ) metabolism and resistance in epilepsy patients, but the relevance remains controversial. To reveal the relationships among the gene polymorphisms of ABCB1, EPHX1, SCN1A and the metabolism and resistance of CBZ, the databases of PubMed, EMBASE, Cochrane Library, Chinese National Knowledge Infrastructure, Chinese Science and Technique Journals, China Biology medicine disc and Wan Fang were retrieved for suitable studies up to April 2021. 18 studies containing 3293 epilepsy patients were included. The result revealed the gene polymorphism of ABCB1 c.3435C > T is significantly associated with altered concentration-dose ratios of CBZ (CDRCBZ) (CC vs. CT, OR = 0.25 (95% CI: 0.08-0.42), P = 0.004), and EPHX c.416A > G gene polymorphism may also significantly adjusted the concentration-dose ratios of carbamazepine-10, 11-trans dihydrodiol (CDRCBZD) (AA vs. GG, OR = 0.48 (95% CI: 0.01-0.96), P = 0.045; AG vs. GG, OR = 0.68 (95% CI: 0.16-1.20), P = 0.010, respectively) and the ratio of CBZD:carbamazepine-10,11-epoxide (CBZE) (CDRCBZD:CDRCBZE) (AG vs GG, OR = 0.83 (95% CI: 0.31-1.36), P = 0.002). Furthermore, ABCB1 c.3435C > T polymorphism was also observed to be significantly influenced CBZ resistance (CC vs TT, OR = 1.78 (95% CI: 1.17-2.72), P = 0.008; CT vs TT, OR = 1.60 (95% CI: 1.12-2.30), P = 0.01; CC + CT vs TT, OR = 1.61 (95% CI: 1.15-2.26), P = 0.006, respectively). Therefore, CBZ metabolism and resistance in patients with epilepsy may be adjusted by the gene polymorphisms of ABCB1 c.3435C > T and EPHX1 c.416A > G which provides the further scientific basis for clinical individualized therapy of epilepsy. However, larger sample size studies are still needed to provide further conclusive evidence.

Quantitative MRI susceptibility mapping reveals cortical signatures of changes in iron, calcium and zinc in malformations of cortical development in children with drug-resistant epilepsy.

OBJECTIVE: Malformations of cortical development (MCD), including focal cortical dysplasia (FCD), are the most common cause of drug-resistant focal epilepsy in children. Histopathological lesion characterisation demonstrates abnormal cell types and lamination, alterations in myelin (typically co-localised with iron), and sometimes calcification. Quantitative susceptibility mapping (QSM) is an emerging MRI technique that measures tissue magnetic susceptibility (χ) reflecting it's mineral composition. We used QSM to investigate abnormal tissue composition in a group of children with focal epilepsy with comparison to effective transverse relaxation rate (R2*) and Synchrotron radiation X-ray fluorescence (SRXRF) elemental maps. Our primary hypothesis was that reductions in χ would be found in FCD lesions, resulting from alterations in their iron and calcium content. We also evaluated deep grey matter nuclei for changes in χ with age. METHODS: QSM and R2* maps were calculated for 40 paediatric patients with suspected MCD (18 histologically confirmed) and 17 age-matched controls. Patients' sub-groups were defined based on concordant electro-clinical or histopathology data. Quantitative investigation of QSM and R2* was performed within lesions, using a surface-based approach with comparison to homologous regions, and within deep brain regions using a voxel-based approach with regional values modelled with age and epilepsy as covariates. Synchrotron radiation X-ray fluorescence (SRXRF) was performed on brain tissue resected from 4 patients to map changes in iron, calcium and zinc and relate them to MRI parameters. RESULTS: Compared to fluid-attenuated inversion recovery (FLAIR) or T1-weighted imaging, QSM improved lesion conspicuity in 5% of patients. In patients with well-localised lesions, quantitative profiling demonstrated decreased χ, but not R2*, across cortical depth with respect to the homologous regions. Contra-lateral homologous regions additionally exhibited increased χ at 2-3 mm cortical depth that was absent in lesions. The iron decrease measured by the SRXRF in FCDIIb lesions was in agreement with myelin reduction observed by Luxol Fast Blue histochemical staining. SRXRF analysis in two FCDIIb tissue samples showed increased zinc and calcium in one patient, and decreased iron in the brain region exhibiting low χ and high R2* in both patients. QSM revealed expected age-related changes in the striatum nuclei, substantia nigra, sub-thalamic and red nucleus. CONCLUSION: QSM non-invasively revealed cortical/sub-cortical tissue alterations in MCD lesions and in particular that χ changes in FCDIIb lesions were consistent with reduced iron, co-localised with low myelin and increased calcium and zinc content. These findings suggest that measurements of cortical χ could be used to characterise tissue properties non-invasively in epilepsy lesions.