Mouse Model Of Status Epilepticus Research Articles

(+)-Borneol inhibits neuroinflammation and M1 phenotype polarization of microglia in epileptogenesis through the TLR4-NFκB signaling pathway

ObjectiveTo investigate the effect of (+)-borneol on neuroinflammation and microglia phenotype polarization in epileptogenesis and its possible mechanism.MethodsBased on mouse models of status epilepticus (SE) induced by pilocarpine, and treated with 15 mg/kg (+)-borneol, western-blot was used to detect the expressions of NeuN, Iba-1, TLR4, p65 and p-p65 in the hippocampus. Immunofluorescence was used to detect the expression of apoptosis-related proteins Bax and Bcl-2. To explore the effect of (+)-borneol on microglia in vitro, we used the kainic acid-induced microglia model and the concentration of (+)-borneol was 25 μM according to CCK-8 results. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-10 (IL-10) in the supernatant of each group was detected by ELISA. The nitric oxide (NO) content in the supernatant was detected by Griess method. The expressions of Iba-1 and TLR4-NFκB signaling pathway-related proteins (TLR4, p65, p-p65) were detected by Western-Blot. Immunofluorescence was used to detect microglia’s M1 and M2 phenotype polarization and the expression of Iba-1 and TLR4.Results(+)-borneol reduced hippocampal neuronal injury, apoptosis, and microglia activation by inhibiting the TLR-NFκB signaling pathway in SE mice. TLR4 agonist LPS partially reversed the neuroprotective effect of (+)-borneol. In the KA-induced microglia model, (+)-borneol inhibited microglia activation, M1 phenotype polarization, and secretion of pro-inflammatory cytokines through the TLR4-NFκB signaling pathway. LPS treatment inhibited the therapeutic effects of (+)-borneol.Conclusion(+)-borneol inhibits microglial neuroinflammation and M1 phenotype polarization through TLR4-NFκB signaling pathway and reduces neuronal damage and apoptosis in SE mice. Therefore, (+)-borneol may be a potential drug for epilepsy modification therapy.

Wogonin mitigates microglia-mediated synaptic over-pruning and cognitive impairment following epilepsy

BackgroundEpilepsy, a neurological disorder characterized by recurrent abnormal neuronal discharges, leading to brain dysfunction and imposing significant psychological and economic burdens on patients. Microglia, the resident immune cells within the central nervous system (CNS), play a crucial role in maintaining CNS homeostasis. However, activated microglia can excessively prune synapses, exacerbating neuronal damage and cognitive dysfunction following epilepsy. Wogonin, a flavonoid from Scutellaria Baicalensis, has known neuroprotective effects via anti-inflammatory and antioxidative mechanisms, but its impact on microglial activation and synaptic pruning in neurons post-epilepsy remains unclear. MethodsSynaptic density was assessed using presynaptic marker Synaptophysin and postsynaptic marker Psd-95, and microglial phagocytosis was evaluated with fluorescent microspheres. Pilocarpine-induced mouse model of status epilepticus was used to evaluate synaptic density changes of mouse hippocampus following an intraperitoneal injection of wogonin (50 and 100 mg/kg). Memory and cognitive function in mice were subsequently evaluated using the Y-maze, object recognition, and Morris water maze tests. Single-cell sequencing was employed to investigate the underlying causes of microglial state alterations, followed by experimental validation. ResultsMicroglia were transitioned to an activated state post-epilepsy, exhibiting significantly enhanced phagocytic capacity. Correspondingly, levels of synaptophysin and Psd-95 were markedly reduced in neurons. Treatment with wogonin (100 mg/kg) significantly increased neuronal synaptic density and improved learning and memory deficits in epileptic mice. Further investigation revealed that wogonin inhibits the release of pro-inflammatory cytokines and synaptic phagocytosis of microglia by activating the AKT/FoxO1 pathway. ConclusionsWogonin could alleviate excessive synaptic pruning of epileptic neurons by microglia and improve cognitive dysfunction of epileptic mice via the AKT/FoxO1 pathway.

Spatial and amplitude dynamics of neurostimulation: Insights from the acute intrahippocampal kainate seizure mouse model.

Neurostimulation is an emerging treatment for patients with drug-resistant epilepsy, which is used to suppress, prevent, and terminate seizure activity. Unfortunately, after implantation and despite best clinical practice, most patients continue to have persistent seizures even after years of empirical optimization. The objective of this study is to determine optimal spatial and amplitude properties of neurostimulation in inhibiting epileptiform activity in an acute hippocampal seizure model. We performed high-throughput testing of high-frequency focal brain stimulation in the acute intrahippocampal kainic acid mouse model of status epilepticus. We evaluated combinations of six anatomic targets and three stimulus amplitudes. We found that the spike-suppressive effects of high-frequency neurostimulation are highly dependent on the stimulation amplitude and location, with higher amplitude stimulation being significantly more effective. Epileptiform spiking activity was significantly reduced with ipsilateral 250 μA stimulation of the CA1 and CA3 hippocampal regions with 21.5% and 22.2% reductions, respectively. In contrast, we found that spiking frequency and amplitude significantly increased with stimulation of the ventral hippocampal commissure. We further found spatial differences with broader effects from CA1 versus CA3 stimulation. These findings demonstrate that the effects of therapeutic neurostimulation in an acute hippocampal seizure model are highly dependent on the location of stimulation and stimulus amplitude. We provide a platform to optimize the anti-seizure effects of neurostimulation, and demonstrate that an exploration of the large electrical parameter and location space can improve current modalities for treating epilepsy. In this study, we tested how electrical pulses in the brain can help control seizures in mice. We found that the electrode's placement and the stimulation amplitude had a large effect on outcomes. Some brain regions, notably nearby CA1 and CA3, responded positively with reduced seizure-like activities, while others showed increased activity. These findings emphasize that choosing the right spot for the electrode and adjusting the strength of electrical pulses are both crucial when considering neurostimulation treatments for epilepsy.

Mice deficient in complement C3 are protected against recognition memory deficits and astrogliosis induced by status epilepticus.

Status epilepticus (SE) can significantly increase the risk of temporal lobe epilepsy (TLE) and cognitive comorbidities. A potential candidate mechanism underlying memory defects in epilepsy may be the immune complement system. The complement cascade, part of the innate immune system, modulates inflammatory and phagocytosis signaling, and has been shown to contribute to learning and memory dysfunctions in neurodegenerative disorders. We previously reported that complement C3 is elevated in brain biopsies from human drug-resistant epilepsy and in experimental rodent models. We also found that SE-induced increases in hippocampal C3 levels paralleled the development of hippocampal-dependent spatial learning and memory deficits in rats. Thus, we hypothesized that SE-induced C3 activation contributes to this pathophysiology in a mouse model of SE and acquired TLE. In this study C3 knockout (KO) and wild type (WT) mice were subjected to one hour of pilocarpine-induced SE or sham conditions (control; C). Following a latent period of two weeks, recognition memory was assessed utilizing the novel object recognition (NOR) test. Western blotting was utilized to determine the protein levels of C3 in hippocampal lysates. In addition, we assessed the protein levels and distribution of the astrocyte marker glial fibrillary acidic protein (GFAP). In the NOR test, control WT + C or C3 KO + C mice spent significantly more time exploring the novel object compared to the familiar object. In contrast, WT+SE mice did not show preference for either object, indicating a memory defect. This deficit was prevented in C3 KO + SE mice, which performed similarly to controls. In addition, we found that SE triggered significant increases in the protein levels of GFAP in hippocampi of WT mice but not in C3 KO mice. These findings suggest that ablation of C3 prevents SE-induced recognition memory deficits and that a C3-astrocyte interplay may play a role. Therefore, it is possible that enhanced C3 signaling contributes to SE-associated cognitive decline during epileptogenesis and may serve as a potential therapeutic target for treating cognitive comorbidities in acquired TLE.

Multi-proteomic Analysis Revealed Distinct Protein Profiles in Cerebrospinal Fluid of Patients Between Anti-NMDAR Encephalitis NORSE and Cryptogenic NORSE.

New-onset refractory status epilepticus (NORSE) is rare but intractable. Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis and cryptogenic etiologies are the two major causes of NORSE with distinct clinical features. To elucidate the underlying mechanisms, 6 patients with anti-NMDAR encephalitis NORSE and 5 with cryptogenic NORSE (C-NORSE) were enrolled. Five patients of cerebrovascular disorders were used as controls. Quantitative proteomic analysis of the cerebrospinal fluid (CSF) samples of the patients revealed 101 and 56 proteins were changed, respectively. The average fold-change of the upregulated proteins, namely up-proteomic score in this study, was positively correlated with the severity and prognosis of the diseases, including ICU stay (r = 0.9308, P = 0.0035 in NMDAR group; r = 0.8977, P = 0.0193 in C-NORSE group), mRS score at discharge (r = 0.9710, P = 0.0111 in NMDAR group; r = 0.7071, P = 0.2000 in C-NORSE group), and time taken for patients awaking from a coma (r = 0.8823, P = 0.0100 in NMDAR group; r = 0.7906, P = 0.2000 in C-NORSE group). Pathways involved in humoral immune response, wound healing, and epigenetic regulation of transcription were upregulated in anti-NMDAR encephalitis NORSE. Pathways of innate and lymphocyte mediated immune response, synaptic functions, ubiquitination, and cell apoptosis were up-regulated in C-NORSE, which was consistent with a mouse model of status epilepticus. Fc receptor and B cell mediated immunity signaling pathways were downregulated in C-NORSE. Immunome microarray analysis demonstrated high autoantibody targeting 48 proteins in CSF samples of anti-NMDAR encephalitis NORSE. While the reaction was kept at a very low level in C-NORSE. There is no significant difference in inflammatory cytokine level between each group. The level of IL-4 (r = 0.7435, P = 0.0451), IL-13 (r = 0.7643, P = 0.0384), IFN-γ (r = 0.7973, P = 0.0287) and TNF-α (r = 0.8598, P = 0.0141) in NMDAR group, and IL-6 (r = 0.8479, P = 0.0348), IL-8 (r = 0.9076, P = 0.0166) in C-NORSE group were positively correlated with the up-proteomic score. The present study suggests that the up-proteomic score of CSF could be a promising indicator for assessment of the severity of anti-NMDAR encephalitis NORSE and C-NORSE. The distinct CSF proteomes imply different pathogenic mechanisms of the two diseases, and immunotherapy strategies as well.

Mesial Temporal Lobe Epilepsy (MTLE) Drug-Refractoriness Is Associated With P2X7 Receptors Overexpression in the Human Hippocampus and Temporal Neocortex and May Be Predicted by Low Circulating Levels of miR-22.

Objective: ATP-gated ionotropic P2X7 receptors (P2X7R) actively participate in epilepsy and other neurological disorders. Neocortical nerve terminals of patients with Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS) express higher P2X7R amounts. Overexpression of P2X7R bolsters ATP signals during seizures resulting in glial cell activation, cytokines production, and GABAergic rundown with unrestrained glutamatergic excitation. In a mouse model of status epilepticus, increased expression of P2X7R has been associated with the down-modulation of the non-coding micro RNA, miR-22. MiR levels are stable in biological fluids and normally reflect remote tissue production making them ideal disease biomarkers. Here, we compared P2X7R and miR-22 expression in epileptic brains and in the serum of patients with MTLE-HS, respectively.Methods: Quantitative RT-PCR was used to evaluate the expression of P2X7R in the hippocampus and anterior temporal lobe of 23 patients with MTLE-HS and 10 cadaveric controls. Confocal microscopy and Western blot analysis were performed to assess P2X7R protein amounts. MiR-22 expression was evaluated in cell-free sera of 40 MTLE-HS patients and 48 healthy controls.Results: Nerve terminals of the hippocampus and neocortical temporal lobe of MTLE-HS patients overexpress (p < 0.05) an 85 kDa P2X7R protein whereas the normally occurring 67 kDa receptor protein dominates in the brain of the cadaveric controls. Contrariwise, miR-22 serum levels are diminished (p < 0.001) in MTLE-HS patients compared to age-matched control blood donors, a situation that is more evident in patients requiring multiple (>3) anti-epileptic drug (AED) regimens.Conclusion: Data show that there is an inverse relationship between miR-22 serum levels and P2X7R expression in the hippocampus and neocortex of MTLE-HS patients, which implies that measuring serum miR-22 may be a clinical surrogate of P2X7R brain expression in the MTLE-HS. Moreover, the high area under the ROC curve (0.777; 95% CI 0.629–0.925; p = 0.001) suggests that low miR-22 serum levels may be a sensitive predictor of poor response to AEDs among MTLE-HS patients. Results also anticipate that targeting the miR-22/P2X7R axis may be a good strategy to develop newer AEDs.

Modulation of Microglia M2 Polarization and Alleviation of Hippocampal Neuron Injury By MiR-106b-5p/RGMa in a Mouse Model of Status Epilepticus.

MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level. The miRNA miR-106b-5p has been linked to epilepsy, but its specific role and mechanism of action remain unclear. This was investigated in the present study using a mouse model of pilocarpine-induced status epilepticus and an in vitro system of HT22 hippocampal cells treated with Mg2+-free solution and cocultured with BV2 microglia cells. We found that inhibiting miR-106b-5p expression promoted microglia M2 polarization, reduced the inflammatory response, and alleviated neuronal injury. These effects involved modulation of the repulsive guidance molecule A (RGMa)-Rac1-c-Jun N-terminal kinase (JNK)/p38-mitogen-activated protein kinase (MAPK) signaling axis. Our results suggest that therapeutic strategies targeting miR-106b-5p or downstream factors can be effective in preventing epileptogenesis or treating epilepsy.

A Comparison of Epileptogenic Effect of Status Epilepticus Treated With Diazepam, Midazolam, and Pentobarbital in the Mouse Pilocarpine Model of Epilepsy

Status epilepticus (SE) is a medical emergency associated with acute severe systemic damage and high mortality. Moreover, symptomatic SE is one of the highest risk factors for epileptogenesis. While the antiepileptic drugs (AEDs) are chosen in favor of acute control of SE, the potential short-term and long-term effects of such AEDs have been ignored in clinics. In this study, we hypothesized that AEDs that are used to control acute SE might affect the feasibility for the chronic development of epileptogenesis after SE. Therefore, we sought to compare the epileptogenic effects of SE that are terminated by three AEDs, i.e., diazepam, midazolam, and pentobarbital, which are widely used as first-line anti-SE AEDs. For this purpose, we used a mouse model of SE induced by intraperitoneal (i.p.) injection of lithium chloride (LiCl)-pilocarpine. The pilocarpine-induced SE was terminated with diazepam, midazolam, or pentobarbital. Then we compared short-term and long-term effects of SE with different AED treatments by examining SE-associated mortality and behavioral spontaneous recurrent seizures (SRSs) and by using magnetic resonance imaging (MRI) and immunohistochemistry to evaluate pathological and cellular alterations of mice in the different treatment groups. We found that i.p. injections of diazepam (5 mg/kg), midazolam (10 mg/kg), and pentobarbital (37.5 mg/kg) were able to terminate acute pilocarpine-SE effectively, while pentobarbital treatment showed less neuroprotective action against lethality in the short phase following SE. Long-term evaluation following SE revealed that SE treated with midazolam had resulted in relatively less behavioral SRS, less hippocampal atrophy, and milder neuronal loss and gliosis. Our data revealed an obvious advantage of midazolam vs. diazepam or pentobarbital in protecting the brain from epileptogenesis. Therefore, if midazolam provides as strong action to quench SE as other AEDs in clinics, midazolam should be the first choice of anti-SE AEDs as it provides additional benefits against epileptogenesis.

Neuronal circuits sustaining neocortical-injury-induced status epilepticus

ObjectivesAcute injuries or insults to the cortex, such as trauma, subarachnoid hemorrhage, lobar hemorrhage, can cause seizures or status epilepticus(SE). Neocortical SE is associated with coma, worse prognosis, delayed recovery, and the development of epilepsy. The anatomical structures progressively recruited during neocortical-onset status epilepticus (SE) is unknown. Therefore, we constructed large-scale maps of brain regions active during neocortical SE. MethodsWe used a neocortical injury-induced SE mouse model. We implanted cobalt (Co) in the right supplementary motor cortex (M2). We 16 h later administered a homocysteine injection (845 mg/kg, intraperitoneal) to C57Bl/6 J mice to induce SE and monitored it by video and EEG. We harvested animals for 1 h (early-stage) and 2 h (late-stage) following homocysteine injections. To construct activation maps, we immunolabeled whole-brain sections for cFos and NeuN, imaged them using a confocal microscope and quantified cFos immunoreactivity (IR). ResultsSE in the early phase consisted of discrete, focal intermittent seizures, which became continuous and bilateral in the late stage. In this early stage, cFos IR was primarily observed in the right hemisphere, ipsilateral to the Co lesion, specifically in the motor cortex, retrosplenial cortex, somatosensory cortex, anterior cingulate cortex, lateral and medial septal nuclei, and amygdala. We observed bilateral cFos IR in brain regions during the late stage, indicating the bilateral spread of focal seizures. We found increased cFOS IR in the bilateral somatosensory cortex and the motor cortex and subcortical regions, including the amygdala, thalamus, and hypothalamus. There was noticeably different, intense cFos IR in the bilateral hippocampus compared to the early stage. In addition, there was higher activity in the cortex ipsilateral to the seizure focus during the late stage compared with the early one. ConclusionWe present a large-scale, high-resolution map of seizure spread during neocortical injury-induced SE. Cortico-cortical and cortico subcortical re-entrant circuits sustain neocortical SE. Neuronal loss following neocortical SE, distant from the neocortical focus, may result from seizures.

HOP protein expression in the hippocampal dentate gyrus is acutely downregulated in a status epilepticus mouse model.

Status epilepticus (SE) is a neurological emergency, and delayed management can lead to higher morbidity and mortality. It is thought that prolonged seizures stimulate stem cells in the hippocampus and that epileptogenesis may arise from aberrant connections formed by newly born cells, while others have suggested that the acute neuroinflammation and gliosis often seen in epileptic hippocampi contribute to hyperexcitability and epilepsy development. Previous studies have identified the expression of homeodomain-only protein (HOP) in the hippocampal dentate gyrus (HDG) and the heart. HOP was found to be a regulator of cell proliferation and differentiation during heart development, while it maintains the ‘heart conduction system’ in adulthood. However, little is known about HOP function in the adult HDG, particularly in the SE setting. Here, a HOP immunohistochemical profile in an SE mouse model was established. A total of 24 adult mice were analyzed 3–10 days following the SE episode, the ‘acute phase’. Our findings demonstrate a significant downregulation of HOP and BLBP protein expression in the SE group following SE episodes, while HOP/Ki67 coexpression did not remarkably differ. Furthermore, coexpression of HOP/S100β and HOP/Prox1 was not observed, although we noticed insignificant HOP/DCX coexpression level. The findings of this study show no compelling evidence of proliferation, and newly added neurons were not identified during the acute phase following SE, although HOP protein expression was significantly decreased in the HDG. Similar to its counterpart in the adult heart, this suggests that HOP seems to play a key role in regulating signal conduction in adult hippocampus. Moreover, acute changes in HOP expression following SE could be part of an inflammatory response that could subsequently influence epileptogenicity.

Persistent neuropathology and behavioral deficits in a mouse model of status epilepticus induced by acute intoxication with diisopropylfluorophosphate

Organophosphate (OP) nerve agents and pesticides are a class of neurotoxic compounds that can cause status epilepticus (SE), and death following acute high-dose exposures. While the standard of care for acute OP intoxication (atropine, oxime, and high-dose benzodiazepine) can prevent mortality, survivors of OP poisoning often experience long-term brain damage and cognitive deficits. Preclinical studies of acute OP intoxication have primarily used rat models to identify candidate medical countermeasures. However, the mouse offers the advantage of readily available knockout strains for mechanistic studies of acute and chronic consequences of OP-induced SE. Therefore, the main objective of this study was to determine whether a mouse model of acute diisopropylfluorophosphate (DFP) intoxication would produce acute and chronic neurotoxicity similar to that observed in rat models and humans following acute OP intoxication. Adult male C57BL/6J mice injected with DFP (9.5 mg/kg, s.c.) followed 1 min later with atropine sulfate (0.1 mg/kg, i.m.) and 2-pralidoxime (25 mg/kg, i.m.) developed behavioral and electrographic signs of SE within minutes that continued for at least 4 h. Acetylcholinesterase inhibition persisted for at least 3 d in the blood and 14 d in the brain of DFP mice relative to vehicle (VEH) controls. Immunohistochemical analyses revealed significant neurodegeneration and neuroinflammation in multiple brain regions at 1, 7, and 28 d post-exposure in the brains of DFP mice relative to VEH controls. Deficits in locomotor and home-cage behavior were observed in DFP mice at 28 d post-exposure. These findings demonstrate that this mouse model replicates many of the outcomes observed in rats and humans acutely intoxicated with OPs, suggesting the feasibility of using this model for mechanistic studies and therapeutic screening.

MiR-106b-5p upregulation is associated with microglial activation and inflammation in the mouse hippocampus following status epilepticus.

To investigate the association of miR-106b-5p with neuroinflammation and microglial activation in a status epilepticus (SE) mouse model. We examined changes in the expression of microRNA-106b-5p (miRNA-106b-5p), repulsive guidance molecule A (RGMa), triggering receptor expressed on myeloid cells 2 (TREM2), and the microglia-related markers interleukin (IL)-1β, IL-4, IL-6, IL-10, inducible nitric oxide synthase (iNOS), and arginase-1 (Arg-1) in the mouse hippocampus of the lithium-pilocarpine-induced SE mouse model. Eighty-four female C57BL/6 mice were randomly divided into a normal control group (n = 12), and six SE groups (n = 12/group), which were monitored at 6h and at 1, 3, 7, 14, and 21days (d) post-SE induction. Unlike in the dentate gyrus, immunohistochemical staining revealed prominent neuronal swelling at 6h, significant neuronal loss and apoptosis on day 3, and recovery by day 14 in the hippocampal cornu ammonis (CA)1 and CA3 pyramidal cells in SE mice. We noted elevated levels of miRNA-106b-5p and all microglia-related markers, which peaked at 3days post-SE, except IL-4, which peaked at 7days post-SE, indicating inflammation and microglial activation. RGMa and TREM2 levels decreased at 6h post-SE. All markers but miRNA-106b-5p, RGMa, and TREM2 returned to baseline levels at 21days post-SE. Dual luciferase reporter gene assay showed that microRNA-106b-5p can interact with RGMa. We observed that miR-106b-5p level increased while both RGMa and TREM2 levels decreased post-SE and showed associations with microglial activation and inflammation in the mouse hippocampus, suggesting their potential as SE therapeutic targets.

Complement C3 Aggravates Post-epileptic Neuronal Injury Via Activation of TRPV1.

Epilepsy is a brain condition characterized by the recurrence of unprovoked seizures. Recent studies have shown that complement component 3 (C3) aggravate the neuronal injury in epilepsy. And our previous studies revealed that TRPV1 (transient receptor potential vanilloid type 1) is involved in epilepsy. Whether complement C3 regulation of neuronal injury is related to the activation of TRPV1 during epilepsy is not fully understood. We found that in a mouse model of status epilepticus (SE), complement C3 derived from astrocytes was increased and aggravated neuronal injury, and that TRPV1-knockout rescued neurons from the injury induced by complement C3. Circular RNAs are abundant in the brain, and the reduction of circRad52 caused by complement C3 promoted the expression of TRPV1 and exacerbated neuronal injury. Mechanistically, disorders of neuron-glia interaction mediated by the C3-TRPV1 signaling pathway may be important for the induction of neuronal injury. This study provides support for the hypothesis that the C3-TRPV1 pathway is involved in the prevention and treatment of neuronal injury and cognitive disorders.

The complement C3-C3aR pathway mediates microglia-astrocyte interaction following status epilepticus.

Gliosis is a histopathological characteristic of epilepsy that comprises activated microglia and astrocytes. It is unclear whether or how crosstalk occurs between microglia and astrocytes in the evolution of epilepsy. Here, we report in a mouse model of status epilepticus, induced by intracerebroventricular injection of kainic acid (KA), sequential activation of microglia and astrocytes and their close spatial interaction in the hippocampal CA3 region. Microglial ablation reduced astrocyte activation and their upregulation of complement C3. When compared to wild-type mice, both C3-/- and C3aR-/- mice had significantly less microglia-astrocyte interaction in response to KA-induced status epilepticus. Additionally, KA-injected C3-/- mice had significantly less histochemical evidence of neurodegeneration. The results suggest that the C3-C3aR pathway contributes to KA-induced neurodegeneration by mediating microglia-astrocyte communication. The C3-C3aR pathway may prove to be a potential therapeutic target for epilepsy treatment.

Knockout of Transient Receptor Potential Melastatin 4 Channel Mitigates Cerebral Edema and Neuronal Injury After Status Epilepticus in Mice.

This study aimed to evaluate whether the knockout of transient receptor potential melastatin 4 (TRPM4) could reduce cerebral edema and improve neurologic outcome in a mouse model of status epilepticus (SE). Wild-type (WT) (n = 61) and Trpm4-/- mice (n = 61) with behavioral seizures induced by lithium (10 mEq/kg) and pilocarpine (30-40 mg/kg) were terminated 2.5 hours after the onset of SE. After SE, 28 WT-SE and 27 Trpm4-/--SE mice were observed for 28 days and assessed for survival and cognitive function; the others were killed after 24 hours, 72 hours, or 7 days, and evaluated for cerebral edema and histological injury. In comparison to WT-SE mice, the mortality and cognitive deficit for Trpm4-/--SE mice following SE after 28 days were significantly ameliorated. Trpm4-/--SE mice also showed less water content and cerebral edema assessed by magnetic resonance imaging, and decreased blood-brain barrier breakdown after SE. Moreover, Trpm4 deficiency significantly mitigated neuronal loss, cellular necrosis and apoptosis in the hippocampus and piriform cortex and mitigated astrocytosis and microgliosis. In conclusion, this study suggests that Trmp4 may represent a new target for improving outcomes after SE.

Antagomirs targeting miR-142–5p attenuate pilocarpine-induced status epilepticus in mice

MicroRNAs (miRNAs) are reported to involve in pathogenesis of temporal lobe epilepsy (TLE). miR-142–5p is found increased in TLE, but its role remains unknown. In the study, we established a mouse model of status epilepticus (SE) with pilocarpine and a cell model of TLE. Quantitative real-time PCR revealed an up-regulation of miR-142–5p and down-regulation of mitochondrial Rho 1 (Miro1) in the mouse mode of SE. Administration of miR-142–5p antagomirs via intracerebroventricular injection attenuated pilocarpine-induced SE and hippocampal damage, and alleviated mitochondrial dysfunction along with increased mitochondrial membrane potential and intracellular ATP and Ca (2+) levels. The expression of mitochondrial trafficking kinesin protein (Trak) 1 and Trak2 was up-regulated by inhibiting miR-142–5p. Antagomirs targeting miR-142–5p suppressed pilocarpine-induced oxidative stress as evidenced by decreased ROS generation and MPO activity, and increased SOD activity. Silencing miR-142–5p reduced neuronal death in pilocarpine-treated hippocampus and magnesium-free (MGF)-treated neurons. Inhibition of miR-142–5p decreased cytoplasmic Cytochrome C and increased mitochondrial Cytochrome C, reduced cleaved-caspase3 and Bax levels, and elevated Bcl2 in vivo and in vitro. Further, dual-luciferase assay verified Miro1 as a target of miR-142–5p, suggesting that miR-142–5p might function via targeting Mrio1. Depletion of Miro1 inhibited the protective effect of silencing miR-142–5p on hippocampal neurons in vitro. Taken together, down-regulation of miR-142–5p via targeting Miro1 inhibits neuronal death and mitochondrial dysfunction, and thus attenuates pilocarpine-induced SE, suggesting the potential involvement of miR-142–5p in the pathogenesis of TLE.

Heparin ameliorates cerebral edema and improves outcomes following status epilepticus by protecting endothelial glycocalyx in mice

Blood brain barrier (BBB) hyperpermeability and brain edema contribute to increased seizure susceptibility and brain injury in status epilepticus (SE). The endothelial glycocalyx is the coating on luminal side of the endothelium and can be considered as the first barrier of BBB. Currently, little is known about the effects of endothelial glycocalyx in SE. We hypothesized glycocalyx degradation could be considered as a first step in the pathophysiology of SE. The study aimed to investigate the impacts of glycocalyx integrity loss on brain damage in a C57BL/6 mouse model of SE induced by lithium-pilocarpine and whether heparin, a competitive antagonist against heparinase, improves survival and neurological outcome. Compared to controls, glycocalyx was significantly degraded after SE, which was mitigated by heparin. The glycocalyx disruption was associated with higher BBB permeability and aggravated brain edema at 72 h after SE, as well as lower survival rate and poorer neurologic outcome. Conversely, preservation of glycocalyx by heparin could reduce SE-induced activation of glia cells, BBB leakage, brain edema, decrease the expressions of inflammatory factors and improve neurologic outcome. The study highlights the importance of glycocalyx degradation in cerebral edema and SE outcome, and indicates heparin treatment may be a new strategy for brain protection in SE.

Alteration of Gene Associated with Retinoid-interferon-induced Mortality-19-expressing Cell Types in the Mouse Hippocampus Following Pilocarpine-induced Status Epilepticus

The gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) plays several significant roles in cellular processes, including ATP synthesis, reactive oxygen species formation, and the regulation of glycolytic enzyme activity, which are closely related to the pathophysiological mechanisms of epilepsy. Therefore, we investigated the expression pattern of GRIM-19 in the CA1 area of the hippocampus in 8-week-old male C57BL/6 mice following pilocarpine-induced status epilepticus (SE). Neuronal death in the hippocampal CA1 area was prominently observed at 4 and 7 days after SE, and astrocytes and microglia became progressively activated beginning at 1 day after SE. GRIM-19 immunoreactivity was decreased in the damaged pyramidal cell layer but markedly increased in the stratum radiatum and stratum lacunosum-moleculare of the hippocampus at 4 and 7 days after SE. In addition, the cell types of GRIM-19-expressing cells in the epileptic hippocampus were identified. GRIM-19 was mainly co-localized in neurons but only slightly expressed in glia in the normal hippocampus. Most of the GRIM-19-positive cells induced by SE in the stratum radiatum and stratum lacunosum-moleculare were glial fibrillary acidic protein-expressing reactive astrocytes. Moreover, we observed that both GRIM-19 and pyruvate kinase isozyme M2, a glycolytic enzyme, were highly expressed in reactive astrocytes after SE. These results indicate that expression of GRIM-19 in the hippocampus is mainly observed in neurons under normal conditions but is altered in the SE mouse model as evidenced by its increased expression in reactive astrocytes. The possible role of GRIM-19 in the glycolytic activity of reactive astrocytes is also discussed.

Optimal concentration of necrostatin-1 for protecting against hippocampal neuronal damage in mice with status epilepticus

Hippocampal neurons undergo various forms of cell death after status epilepticus. Necrostatin-1 specifically inhibits necroptosis mediated by receptor interacting protein kinase 1 (RIP1) and RIP3 receptors. However, there are no reports of necroptosis in mouse models of status epilepticus. Therefore, in this study, we investigated the effects of necrostatin-1 on hippocampal neurons in mice with status epilepticus, and, furthermore, we tested different amounts of the compound to identify the optimal concentration for inhibiting necroptosis and apoptosis. A mouse model of status epilepticus was produced by intraperitoneal injection of kainic acid, 12 mg/kg. Different concentrations of necrostatin-1 (10, 20, 40, and 80 μM) were administered into the lateral ventricle 15 minutes before kainic acid injection. Hippocampal damage was assessed by hematoxylin-eosin staining 24 hours after the model was successfully produced. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, western blot assay and immunohistochemistry were used to evaluate the expression of apoptosis-related and necroptosis-related proteins. Necrostatin-1 alleviated damage to hippocampal tissue in the mouse model of epilepsy. The 40 μM concentration of necrostatin-1 significantly decreased the number of apoptotic cells in the hippocampal CA1 region. Furthermore, necrostatin-1 significantly downregulated necroptosis-related proteins (MLKL, RIP1, and RIP3) and apoptosis-related proteins (cleaved-Caspase-3, Bax), and it upregulated the expression of anti-apoptotic protein Bcl-2. Taken together, our findings show that necrostatin-1 effectively inhibits necroptosis and apoptosis in mice with status epilepticus, with the 40 μM concentration of the compound having an optimal effect. The experiments were approved by the Animal Ethics Committee of Fujian Medical University, China (approval No. 2016-032) on November 9, 2016.

Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy.

Extracellular ATP activates inflammatory responses to tissue injury. It is also implicated in establishing lasting network hyperexcitability in the brain by acting upon independent receptor systems. Whereas the fast-acting P2X channels have well-established roles driving neuroinflammation and increasing hyperexcitability, the slower-acting metabotropic P2Y receptors have received much less attention. Recent studies of P2Y1 receptor function in seizures and epilepsy have produced contradictory results, suggesting that the role of this receptor during seizure pathology may be highly sensitive to context. Here, by using male mice, we demonstrate that the metabotropic P2Y1 receptor mediates either proconvulsive or anticonvulsive responses, dependent on the time point of activation in relation to the induction of status epilepticus. P2Y1 deficiency or a P2Y1 antagonist (MRS2500) administered before a chemoconvulsant, exacerbates epileptiform activity, whereas a P2Y1 agonist (MRS2365) administered at this time point is anticonvulsant. When these drugs are administered after the onset of status epilepticus, however, their effect on seizure severity is reversed, with the antagonist now anticonvulsant and the agonist proconvulsant. This result was consistent across two different mouse models of status epilepticus (intra-amygdala kainic acid and intraperitoneal pilocarpine). Pharmacologic P2Y1 blockade during status epilepticus reduces also associated brain damage, delays the development of epilepsy and, when applied during epilepsy, suppresses spontaneous seizures, in mice. Our data show a context-specific role for P2Y1 during seizure pathology and demonstrate that blocking P2Y1 after status epilepticus and during epilepsy has potent anticonvulsive effects, suggesting that P2Y1 may be a novel candidate for the treatment of drug-refractory status epilepticus and epilepsy.SIGNIFICANCE STATEMENT This is the first study to fully characterize the contribution of a metabotropic purinergic P2Y receptor during acute seizures and epilepsy. The findings suggest that targeting P2Y1 may offer a potential novel treatment strategy for drug-refractory status epilepticus and epilepsy. Our data demonstrate a context-specific role of P2Y1 activation during seizures, switching from a proconvulsive to an anticonvulsive role depending on physiopathological context. Thus, our study provides a possible explanation for seemingly conflicting results obtained between studies of different brain diseases where P2Y1 targeting has been proposed as a potential treatment strategy and highlights that the timing of pharmacological interventions is of critical importance to the understanding of how receptors contribute to the generation of seizures and the development of epilepsy.