Immune profile in blood following non-convulsive epileptic seizures in rats

Non-convulsive status epilepticus (NCSE) is a prolonged epileptic seizure with subtle symptoms that may delay clinical diagnosis. Emerging experimental evidence shows brain pathology and epilepsy development following NCSE. New diagnostic/prognostic tools are therefore needed for earlier and better stratification of treatment. Here we examined whether NCSE initiates a peripheral immune response in blood serum from rats that experienced electrically-induced NCSE. ELISA analysis showed an acute transient increase in serum protein levels including interleukin-6 6 h post-NCSE, similar to the immune reaction in the brain. At 4 weeks post-NCSE, when 75% of rats subjected to NCSE had also developed spontaneous seizures, several immune proteins were altered. In particular, markers associated with microglia, macrophages and antigen presenting cells, such as CD68, MHCII, and galectin-3, were increased and the T-cell marker CD4 was decreased in serum compared to both non-stimulated controls and NCSE rats without spontaneous seizures, without correlation to interictal epileptiform activity. Analyses of serum following intracerebral injection of lipopolysaccharide (LPS) showed an acute increase in interleukin-6, but at 4 weeks unaltered levels of MHCII and galectin-3, an increase in CD8 and CD11b and a decrease in CD68. None of the increased serum protein levels after NCSE or LPS could be confirmed in spleen tissue. Our data identifies the possibility to detect peripheral changes in serum protein levels following NCSE, which may be related to the development of subsequent spontaneous seizures

Levetiracetam and N-Cadherin Antibody Alleviate Brain Pathology Without Reducing Early Epilepsy Development After Focal Non-convulsive Status Epilepticus in Rats

Focal non-convulsive status epilepticus (fNCSE) is a neurological condition characterized by a prolonged seizure that may lead to the development of epilepsy. Emerging experimental evidence implicates neuronal death, microglial activation and alterations in the excitatory and inhibitory synaptic balance as key features in the pathophysiology following fNCSE. We have previously reported alterations in the excitatory adhesion molecule N-cadherin in rats with fNCSE originating from the hippocampus that subsequently also develop spontaneous seizures. In this study, fNCSE rats were treated intraperitoneally with the conventional anti-epileptic drug levetiracetam in combination with intraparenchymal infusion of N-cadherin antibodies (Ab) for 4 weeks post-fNCSE. The N-cadherin Ab was infused into the fornix and immunohistochemically N-cadherin Ab-stained neurons were detected within the dorsal hippocampal structures as well as in superjacent somatosensory cortex. Continuous levetiracetam treatment for 4 weeks post-fNCSE reduced microglia activation, including cell numbers and morphological changes, partly decreased neuronal cell loss, and excitatory post-synaptic scaffold protein PSD-95 expression in selective hippocampal structures. The additional treatment with N-cadherin Ab did not reverse neuronal loss, but moderately reduced microglial activation, and further reduced PSD-95 levels in the dentate hilus of the hippocampus. Despite the effects on brain pathology within the epileptic focus, neither monotherapy with systemic levetiracetam nor levetiracetam in combination with local N-cadherin Ab administration, reduced the amount of focal or focal evolving into bilateral convulsive seizures, seizure duration, or interictal epileptiform activity during 1 month of continuous electroenephalogram recordings within the hippocampus after fNCSE. Behavioral tests for spatial memory, anxiety, social interaction and anhedonia did not detect gross behavioral differences between fNCSE rats with or without treatment. The results reveal the refractory features of the present rodent model of temporal lobe epilepsy following fNCSE, which supports its clinical value for further therapeutic studies. We identify the persistent development of epilepsy following fNCSE, in spite of partly reduced brain pathology within the epileptic focus

Nonconvulsive status epilepticus in rats leads to brain pathology

Objective: Status epilepticus (SE) is an abnormally prolonged epileptic seizure that if associated with convulsive motor symptoms is potentially life threatening for a patient. However, 20%-40% of patients with SE lack convulsive events and instead present with more subtle semiology such as altered consciousness and less motor activity. Today, there is no general consensus regarding to what extent nonconvulsive SE (NCSE) is harmful to the brain, which adds uncertainty to stringent treatment regimes. Methods: Here, we evaluated brain pathology in an experimental rat and mouse model of complex partial NCSE originating in the temporal lobes with Western blot analysis, immunohistochemistry, and ex vivo diffusion tensor imaging (DTI). The NCSE was induced by electrical stimulation with intrahippocampal electrodes and terminated with pentobarbital anesthesia. Video-electroencephalographic recordings were performed throughout the experiment. Results: DTI of mice 7 weeks post-NCSE showed no robust long-lasting changes in fractional anisotropy within the hippocampal epileptic focus. Instead, we found pathophysiological changes developing over time when measuring protein levels and cell counts in extracted brain tissue. At 6 and 24 hours post-NCSE in rats, few changes were observed within the hippocampus and cortical or subcortical structures in Western blot analyses of key components of the cellular immune response and synaptic protein expression, while neurodegeneration had started. However, 1 week post-NCSE, both excitatory and inhibitory synaptic protein levels were decreased in hippocampus, concomitant with an excessive microglial and astrocytic activation. At 4 weeks, a continuous immune response in the hippocampus was accompanied with neuronal loss. Levels of the excitatory synaptic adhesion molecule N-cadherin were decreased specifically in rats that developed unprovoked spontaneous seizures (epileptogenesis) within 1 month following NCSE, compared to rats only exhibiting acute symptomatic seizures within 1 week post-NCSE. Significance: These findings provide evidence for a significant brain pathology following NCSE in an experimental rodent model

Immune response in the eye following epileptic seizures

Background: Epileptic seizures are associated with an immune response in the brain. However, it is not known whether it can extend to remote areas of the brain, such as the eyes. Hence, we investigated whether epileptic seizures induce inflammation in the retina. Methods: Adult rats underwent electrically induced temporal status epilepticus, and the eyes were studied 6 h, 1, and 7 weeks later with biochemical and immunohistochemical analyses. An additional group of animals received CX3CR1 antibody intracerebroventricularly for 6 weeks after status epilepticus. Results: Biochemical analyses and immunohistochemistry revealed no increased cell death and unaltered expression of several immune-related cytokines and chemokines as well as no microglial activation, 6 h post-status epilepticus compared to non-stimulated controls. At 1 week, again, retinal cytoarchitecture appeared normal and there was no cell death or micro- or macroglial reaction, apart from a small decrease in interleukin-10. However, at 7 weeks, even if the cytoarchitecture remained normal and no ongoing cell death was detected, the numbers of microglia were increased ipsi- and contralateral to the epileptic focus. The microglia remained within the synaptic layers but often in clusters and with more processes extending into the outer nuclear layer. Morphological analyses revealed a decrease in surveying and an increase in activated microglia. In addition, increased levels of the chemokine KC/GRO and cytokine interleukin-1β were found. Furthermore, macroglial activation was noted in the inner retina. No alterations in numbers of phagocytic cells, infiltrating macrophages, or vascular pericytes were observed. Post-synaptic density-95 cluster intensity was reduced in the outer nuclear layer, reflecting seizure-induced synaptic changes without disrupted cytoarchitecture in areas with increased microglial activation. The retinal gliosis was decreased by a CX3CR1 immune modulation known to reduce gliosis within epileptic foci, suggesting a common immunological reaction. Conclusions: Our results are the first evidence that epileptic seizures induce an immune response in the retina. It has a potential to become a novel non-invasive tool for detecting brain inflammation through the eyes

Disease Modification by Combinatorial Single Vector Gene Therapy : A Preclinical Translational Study in Epilepsy

Gene therapy has been suggested as a plausible novel approach to achieve seizure control in patients with focal epilepsy that do not adequately respond to pharmacological treatment. We investigated the seizure-suppressant potential of combinatorial neuropeptide Y and Y2 receptor single vector gene therapy based on adeno-associated virus serotype 1 (AAV1) in rats. First, a dose-response study in the systemic kainate-induced acute seizure model was performed, whereby the 1012 genomic particles (gp)/mL titer of the vector was selected as an optimal concentration. Second, an efficacy study was performed in the intrahippocampal kainate chronic model of spontaneous recurrent seizures (SRSs), designed to reflect a likely clinical scenario, with magnetic resonance image (MRI)-guided focal unilateral administration of the vector in the hippocampus during the chronic stage of the disease. The efficacy study demonstrated a favorable outcome of the gene therapy, with a 31% responder rate (more than 50% reduction in SRS frequency) and 13% seizure-freedom rate, whereas no such effects were observed in the control animals. The inter-SRS and SRS cluster intervals were also significantly prolonged in the treated group compared to controls. In addition, the SRS duration was significantly reduced in the treated group but not in the controls. This study establishes the SRS-suppressant ability of the single vector combinatorial neuropeptide Y/Y2 receptor gene therapy in a clinically relevant chronic model of epilepsy

Immune response in blood before and after epileptic and psychogenic non-epileptic seizures

Inflammatory processes may provoke epileptic seizures and seizures may promote an immune reaction. Hence, the systemic immune reaction is a tempting diagnostic and prognostic marker in epilepsy. We explored the immune response before and after epileptic and psychogenic non-epileptic seizures (PNES). Serum samples collected from patients with videoEEG-verified temporal or frontal lobe epilepsy (TLE or FLE) or TLE + PNES showed increased interleukin-6 (IL-6) levels in between seizures (interictally), compared to controls. Patients with PNES had no increase in IL-6. The IL-6 levels increased transiently even further within hours after a seizure (postictally) in TLE but not in FLE patients. The postictal to interictal ratio of additionally five immune factors were also increased in TLE patients only. We conclude that immune factors have the potential to be future biomarkers for epileptic seizures and that the heterogeneity among different epileptic and non-epileptic seizures may be disclosed in peripheral blood sampling independent of co-morbidities