A refined kainate (KA) C57BL/6J mouse model of status epilepticus (SE) using a repeated low dose (RLD) of KA (5 mg/kg, intraperitoneal; at 30 min intervals) was compared with the established single high dose (SHD) of KA (20 mg/kg, intraperitoneal) model. In the RLD group, increased duration of convulsive motor seizures (CMS, Racine scale stage ≥3) with a significant reduction in mortality from 21% to 6% and decreased variability in seizure severity between animals/batches were observed when compared to the SHD group. There was a significant increase in the percentage of animals that reached stage-5 seizures (65% versus 96%) in the RLD group. Integrated real-time video-EEG analysis of both groups, using NeuroScore software, revealed stage-specific spikes and power spectral density characteristics. When the seizures progressed from non-convulsive seizures (NCS, stage 1–2) to CMS (stage 3–5), the delta power decreased which was followed by an increase in gamma and beta power. A transient increase in alpha and sigma power marked the transition from NCS to CMS with characteristic ‘high frequency trigger’ spikes on the EEG, which had no behavioral expression. During SE the spike rate was higher in the RLD group than in the SHD group. Overall these results confirm that RLD of KA is a more robust and consistent mouse model of SE than the SHD of KA mouse model.

The C57BL/6J mouse as a model of seizure/epilepsy is challenging due to high mortality and huge variability in response to kainate. We have recently demonstrated that repeated administration of a low dose of kainate by intraperitoneal route can induce severe status epilepticus (SE) with 94% survival rate. In the present study, based on continuous video-EEG recording for 4-18 weeks from epidurally implanted electrodes on the cortex, we demonstrate that this method also induces immediate epileptogenesis (<1-5 days post-SE). This finding was based on identification of two types of spontaneous recurrent seizures; behavioral convulsive seizures (CS) and electrographic nonconvulsive seizures (NCS). The identification of the spontaneous CS, stage 3-5 types, was based on the behaviors (video) that were associated with the EEG characteristics (stage 3-5 epileptiform spikes), the power spectrum, and the activity counts. The electrographic NCS identification was based on the stage 1-2 epileptiform spike clusters on the EEG and their associated power spectrum. Severe SE induced immediate epileptogenesis in all the mice. The maximum numbers of spontaneous CS were observed during the first 4-6 weeks of the SE and they decreased thereafter. Mild SE also induced immediate epileptogenesis in some mice but the CS were less frequent. In both the severe and the mild SE groups, the spontaneous electrographic NCS persisted throughout the 18 weeks observation period, and therefore this could serve as a chronic model for complex seizures. However, unlike rat kainate models, the C57BL/6J mouse kainate model is a unique regressive CS model of epilepsy. Further studies are required to understand the mechanism of recovery from spontaneous CS in this model, which could reveal novel therapeutic targets for epilepsy.

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Approximately 30% of epilepsy patients do not respond to antiepileptic drugs, representing an unmet medical need. There is evidence that neuroinflammation plays a pathogenic role in drug-resistant epilepsy. The high-mobility group box 1 (HMGB1)/TLR4 axis is a key initiator of neuroinflammation following epileptogenic injuries, and its activation contributes to seizure generation in animal models. However, further work is required to understand the role of HMGB1 and its isoforms in epileptogenesis and drug resistance. Using a combination of animal models and sera from clinically well-characterized patients, we have demonstrated that there are dynamic changes in HMGB1 isoforms in the brain and blood of animals undergoing epileptogenesis. The pathologic disulfide HMGB1 isoform progressively increased in blood before epilepsy onset and prospectively identified animals that developed the disease. Consistent with animal data, we observed early expression of disulfide HMGB1 in patients with newly diagnosed epilepsy, and its persistence was associated with subsequent seizures. In contrast with patients with well-controlled epilepsy, patients with chronic, drug-refractory epilepsy persistently expressed the acetylated, disulfide HMGB1 isoforms. Moreover, treatment of animals with antiinflammatory drugs during epileptogenesis prevented both disease progression and blood increase in HMGB1 isoforms. Our data suggest that HMGB1 isoforms are mechanistic biomarkers for epileptogenesis and drug-resistant epilepsy in humans, necessitating evaluation in larger-scale prospective studies.

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Antiepileptogenic agents that prevent the development of epilepsy following a brain insult remain the holy grail of epilepsy therapeutics. We have employed a label-free proteomic approach that allows quantification of large numbers of brain-expressed proteins in a single analysis in the mouse (male C57BL/6J) kainate (KA) model of epileptogenesis. In addition, we have incorporated two putative antiepileptogenic drugs, postsynaptic density protein-95 blocking peptide (PSD95BP or Tat-NR2B9c) and a highly selective inducible nitric oxide synthase inhibitor, 1400W, to give an insight into how such agents might ameliorate epileptogenesis. The test drugs were administered after the induction of status epilepticus (SE) and the animals were euthanized at 7 days, their hippocampi removed, and subjected to LC-MS/MS analysis. A total of | 1379 TSE ET al.

Intracranial electroencephalography (EEG) is commonly used to study epileptogenesis and epilepsy in experimental models. Chronic gliosis and neurodegeneration at the injury site are known to be associated with surgically implanted electrodes in both humans and experimental models. Currently, however, there are no reports on the impact of intracerebral electrodes on proteins in the hippocampus and proinflammatory cytokines in the cerebral cortex and plasma in experimental models. We used an unbiased, label-free proteomics approach to identify the altered proteins in the hippocampus, and multiplex assay for cytokines in the cerebral cortex and plasma of C57BL/6J mice following bilateral surgical implantation of electrodes into the cerebral hemispheres. Seven days following surgery, a repeated low dose kainate (KA) regimen was followed to induce status epilepticus (SE). Surgical implantation of electrodes reduced the amount of KA necessary to induce SE by 50%, compared with mice without surgery. Tissues were harvested 7 days post-SE (i.e., 14 days post-surgery) and compared with vehicle-treated mice. Proteomic profiling showed more proteins (103, 6.8% of all proteins identified) with significantly changed expression (p &lt; 0.01) driven by surgery than by KA treatment itself without surgery (27, 1.8% of all proteins identified). Further, electrode implantation approximately doubled the number of KA-induced changes in protein expression (55, 3.6% of all identified proteins). Further analysis revealed that intracerebral electrodes and KA altered the expression of proteins associated with epileptogenesis such as inflammation (C1q system), neurodegeneration (cystatin-C, galectin-1, cathepsin B, heat-shock protein 25), blood–brain barrier dysfunction (fibrinogen-α, serum albumin, α2 macroglobulin), and gliosis (vimentin, GFAP, filamin-A). The multiplex assay revealed a significant increase in key cytokines such as TNFα, IL-1β, IL-4, IL-5, IL-6, IL-10, IL12p70, IFN-γ, and KC/GRO in the cerebral cortex and some in the plasma in the surgery group. Overall, these findings demonstrate that surgical implantation of depth electrodes alters some of the molecules that may have a role in epileptogenesis in experimental models.