Prophylactic treatment with the c-Abl inhibitor, neurotinib, diminishes neuronal damage and the convulsive state in pilocarpine-induced mice.

The molecular mechanisms underlying seizure generation remain elusive, yet they are crucial for developing effective treatments for epilepsy. The current study shows that inhibiting c-Abl tyrosine kinase prevents apoptosis, reduces dendritic spine loss, and maintains N-methyl-d-aspartate (NMDA) receptor subunit 2B (NR2B) phosphorylated in in vitro models of excitotoxicity. Pilocarpine-induced status epilepticus (SE) in mice promotes c-Abl phosphorylation, and disrupting c-Abl activity leads to fewer seizures, increases latency toward SE, and improved animal survival. Currently, clinically used c-Abl inhibitors are non-selective and have poor brain penetration. The allosteric c-Abl inhibitor, neurotinib, used here has favorable potency, selectivity, pharmacokinetics, and vastly improved brain penetration. Neurotinib-administered mice have fewer seizures and improved survival following pilocarpine-SE induction. Our findings reveal c-Abl kinase activation as a key factor in ictogenesis and highlight the impact of its inhibition in preventing the insurgence of epileptic-like seizures in rodents and humans.

Brain structural changes in alternating hemiplegia of childhood using single-case voxel-based morphometry analysis.

BACKGROUND AND PURPOSE: Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disease caused by ATP1A3 mutations. Using voxel-based morphometry (VBM) analysis, we compared an AHC patient cohort with controls. Additionally, with single-case VBM analysis, we assessed the associations between clinical severity and brain volume in patients with AHC. MATERIALS AND METHODS: To investigate structural brain changes in gray matter (GM) and white matter (WM) volumes between 9 patients with AHC and 20 age-matched controls, VBM analysis was performed using three-dimensional T1-weighted magnetic resonance imaging. Single-case VBM analysis was also performed on nine patients with AHC to investigate the associations between the respective volumes of GM/WM differences and the motor level, cognitive level, and status epilepticus severity in patients with AHC. RESULTS: Compared with controls, patients with AHC showed significant GM volume reductions in both hippocampi and diffuse cerebellum, and there were WM reductions in both cerebral hemispheres. In patients with AHC, cases with more motor dysfunction, the less GM/WM volume of cerebellum was shown. Three of the six cases with cognitive dysfunction showed a clear GM volume reduction in the insulae. Five of the six cases with status epilepticus showed the GM volume reduction in hippocampi. One case had severe status epilepticus without motor dysfunction and showed no cerebellar atrophy. CONCLUSION: With single-case VBM analysis, we could show the association between region-specific changes in brain volume and the severity of various clinical symptoms even in a small sample of subjects.

Expression of Cytokines and Neurodegeneration in the Rat Hippocampus and Cortex in the Lithium-Pilocarpine Model of Status Epilepticus and the Role of Modulation of Endocannabinoid System.

A significant body of evidence shows that neuroinflammation is one of the key processes in the development of brain pathology in trauma, neurodegenerative disorders, and epilepsy. Various brain insults, including severe and prolonged seizure activity during status epilepticus (SE), trigger proinflammatory cytokine release. We investigated the expression of the proinflammatory cytokines interleukin-1ß (Il1b) and interleukin-6 (Il6), and anti-inflammatory fractalkine (Cx3cl1) in the hippocampus, entorhinal cortex, and neocortex of rats 24 h, 7 days, and 5 months after lithium-pilocarpine SE. We studied the relationship between cytokine expression and neuronal death in the hippocampus and evaluated the effect of modulation of endocannabinoid receptors on neuroinflammation and neurodegeneration after SE. The results of the present study showed that inhibition of endocannabinoid CB1 receptors with AM251 early after SE had a transient neuroprotective effect that was absent in the chronic period and did not affect the development of spontaneous seizures after SE. At the same time, AM251 reduced the expression of Il6 in the chronic period after SE. Higher Cx3cl1 levels were found in rats with more prominent hippocampal neurodegeneration.

Eugenol alleviates neuronal damage via inhibiting inflammatory process against pilocarpine-induced status epilepticus.

Neuroinflammation is one of the most common pathological outcomes in various neurological diseases. A growing body of evidence suggests that neuroinflammation plays a pivotal role in the pathogenesis of epileptic seizures. Eugenol is the major phytoconstituent of essential oils extracted from several plants and possesses protective and anticonvulsant properties. However, it remains unclear whether eugenol exerts an anti-inflammatory effect to protect against severe neuronal damage induced by epileptic seizures. In this study, we investigated the anti-inflammatory action of eugenol in an experimental epilepsy model of pilocarpine-induced status epilepticus (SE). To examine the protective effect of eugenol via anti-inflammatory mechanisms, eugenol (200 mg/kg) was administrated daily for three days after pilocarpine-induced SE onset. The anti-inflammatory action of eugenol was evaluated by examining the expression of reactive gliosis, pro-inflammatory cytokines, nuclear factor-κB (NF-κB), and the nucleotide-binding domain leucine-rich repeat with a pyrin-domain containing 3 (NLRP3) inflammasome. Our results showed that eugenol reduced SE-induced apoptotic neuronal cell death, mitigated the activation of astrocytes and microglia, and attenuated the expression of interleukin-1ß and tumor necrosis factor α in the hippocampus after SE onset. Furthermore, eugenol inhibited NF-κB activation and the formation of the NLRP3 inflammasome in the hippocampus after SE. These results suggest that eugenol is a potential phytoconstituent that suppresses the neuroinflammatory processes induced by epileptic seizures. Therefore, these findings provide evidence that eugenol has therapeutic potential for epileptic seizures.

The amygdala lesioning due to status epilepticus - Changes in mechanisms controlling chloride homeostasis.

OBJECTIVE: Amygdala has been demonstrated as one of the brain sites involved in the control of cardiorespiratory functioning. The structural and physiological alterations induced by epileptic activity are also present in the amygdala and reflect functional changes that may be directly associated with a sudden unexpected death. Seizures are always associated with neuronal damage and changes in the expression of cation-chloride cotransporters and Na/K pumps. In this study, the authors aimed to investigate if these changes are present in the amygdala after induction of status epilepticus with pilocarpine, which may be directly correlated with Sudden Unexpected Death in Epilepsy (SUDEP). METHODS: Pilocarpine-treated wistar rats 60 days after Status Epilepticus (SE) were compared with control rats. Amygdala nuclei of brain slices immunostained for NKCC1, KCC2 and α1-Na+/K+-ATPase, were quantified by optical densitometry. RESULTS: The amygdaloid complex of the animals submitted to SE had no significant difference in the NKCC1 immunoreactivity, but KCC2 immunoreactivity reduced drastically in the peri-somatic sites and in the dendritic-like processes. The α1-Na+/K+-ATPase peri-somatic immunoreactivity was intense in the rats submitted to pilocarpine SE when compared with control rats. The pilocarpine SE also promoted intense GFAP staining, specifically in the basolateral and baso-medial nuclei with astrogliosis and cellular debris deposition. INTERPRETATION: The findings revealed that SE induces lesion changes in the expression of KCC2 and α1-Na+/K+-ATPase meaning intense change in the chloride regulation in the amygdaloid complex. These changes may contribute to cardiorespiratory dysfunction leading to SUDEP.

Reactive microglia are the major source of tumor necrosis factor alpha and contribute to astrocyte dysfunction and acute seizures in experimental temporal lobe epilepsy.

Extensive microglia reactivity has been well described in human and experimental temporal lobe epilepsy (TLE). To date, however, it is not clear whether and based on which molecular mechanisms microglia contribute to the development and progression of focal epilepsy. Astroglial gap junction coupled networks play an important role in regulating neuronal activity and loss of interastrocytic coupling causally contributes to TLE. Here, we show in the unilateral intracortical kainate (KA) mouse model of TLE that reactive microglia are primary producers of tumor necrosis factor (TNF)α and contribute to astrocyte dysfunction and severity of status epilepticus (SE). Immunohistochemical analyses revealed pronounced and persistent microglia reactivity, which already started 4 h after KA-induced SE. Partial depletion of microglia using a colony stimulating factor 1 receptor inhibitor prevented early astrocyte uncoupling and attenuated the severity of SE, but increased the mortality of epileptic mice following surgery. Using microglia-specific inducible TNFα knockout mice we identified microglia as the major source of TNFα during early epileptogenesis. Importantly, microglia-specific TNFα knockout prevented SE-induced gap junction uncoupling in astrocytes. Continuous telemetric EEG recordings revealed that during the first 4 weeks after SE induction, microglial TNFα did not significantly contribute to spontaneous generalized seizure activity. Moreover, the absence of microglial TNFα did not affect the development of hippocampal sclerosis but attenuated gliosis. Taken together, these data implicate reactive microglia in astrocyte dysfunction and network hyperexcitability after an epileptogenic insult.

Characterisation of NLRP3 pathway-related neuroinflammation in temporal lobe epilepsy.

OBJECTIVE: Inflammation of brain structures, in particular the hippocampal formation, can induce neuronal degeneration and be associated with increased excitability manifesting as propensity for repetitive seizures. An increase in the abundance of individual proinflammatory molecules including interleukin 1 beta has been observed in brain tissue samples of patients with pharmacoresistant temporal lobe epilepsy (TLE) and corresponding animal models. The NLRP3-inflammasome, a cytosolic protein complex, acts as a key regulator in proinflammatory innate immune signalling. Upon activation, it leads to the release of interleukin 1 beta and inflammation-mediated neurodegeneration. Transient brain insults, like status epilepticus (SE), can render hippocampi chronically hyperexcitable and induce segmental neurodegeneration. The underlying mechanisms are referred to as epileptogenesis. Here, we have tested the hypothesis that distinct NLRP3-dependent transcript and protein signalling dynamics are induced by SE and whether they differ between two classical SE models. We further correlated the association of NLRP3-related transcript abundance with convulsive activity in human TLE hippocampi of patients with and without associated neurodegenerative damage. METHODS: Hippocampal mRNA- and protein-expression of NLRP3 and associated signalling molecules were analysed longitudinally in pilocarpine- and kainic acid-induced SE TLE mouse models. Complementarily, we studied NLRP3 inflammasome-associated transcript patterns in epileptogenic hippocampi with different damage patterns of pharmacoresistant TLE patients that had undergone epilepsy surgery for seizure relief. RESULTS: Pilocarpine- and kainic acid-induced SE elicit distinct hippocampal Nlrp3-associated molecular signalling. Transcriptional activation of NLRP3 pathway elements is associated with seizure activity but independent of the particular neuronal damage phenotype in KA-induced and in human TLE hippocampi. SIGNIFICANCE: These data suggest highly dynamic inflammasome signalling in SE-induced TLE and highlight a vicious cycle associated with seizure activity. Our results provide promising perspectives for the inflammasome signalling pathway as a target for anti-epileptogenic and -convulsive therapeutic strategies. The latter may even applicable to a particularly broad spectrum of TLE patients with currently pharmacoresistant disease.

Modafinil exerts anticonvulsive effects against lithium-pilocarpine-induced status epilepticus in rats: A role for tumor necrosis factor-α and nitric oxide signaling.

BACKGROUND: Status epilepticus (SE) is a continuous episode of seizures which leads to hippocampal neurodegeneration, severe systemic inflammation, and extreme damage to the brain. Modafinil, a psychostimulant and wake-promoting agent, has exerted neuroprotective and anti-inflammatory effects in previous preclinical studies. The aim of this study was to assess effects of modafinil on the lithium-pilocarpine-induced SE rat model and to explore possible involvement of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) pathways in this regard. METHODS: Status epilepticus was provoked by injection of lithium chloride (127 mg/kg, intraperitoneally [i.p]) and pilocarpine (60 mg/kg, i.p.) in rats. Animals received different modafinil doses (50, 75, 100, and 150 mg/kg, i.p.) and SE scores were documented over 3 hours of duration. Moreover, the role of the nitrergic pathway in the effects of modafinil was evaluated by injection of the non-selective NO synthase (NOS) inhibitor L-NG-Nitro arginine methyl ester (L-NAME, 10 mg/kg, i.p.), the selective neuronal NOS inhibitor 7-nitroindazole (30 mg/kg, i.p.), and the selective inducible NOS inhibitor aminoguanidine (100 mg/kg, i.p.) 15 min before saline/vehicle or modafinil. The ELISA method was used to quantify TNF-α and NO metabolite levels in the isolated hippocampus. RESULTS: Modafinil at 100 mg/kg significantly decreased SE scores (P < 0.01). Pre-treatment with L-NAME, 7-nitroindazole, and aminoguanidine significantly reversed the anticonvulsive effects of modafinil. Status epilepticus-induced animals showed significantly higher NO metabolite and TNF-α levels in their hippocampal tissues, an effect that was reversed by modafinil (100 mg/kg, i.p.) treatment. Administration of NOS inhibitors resulted in excessive NO level reduction but an escalation of TNF-α level in modafinil-treated SE-animals. CONCLUSION: Our study revealed anticonvulsive effects of modafinil in the lithium-pilocarpine-induced SE rat model via possible involvement of TNF-α and nitrergic pathways.

Human Stem Cell-Derived GABAergic Interneurons Establish Efferent Synapses onto Host Neurons in Rat Epileptic Hippocampus and Inhibit Spontaneous Recurrent Seizures.

Epilepsy is a complex disorder affecting the central nervous system and is characterised by spontaneously recurring seizures (SRSs). Epileptic patients undergo symptomatic pharmacological treatments, however, in 30% of cases, they are ineffective, mostly in patients with temporal lobe epilepsy. Therefore, there is a need for developing novel treatment strategies. Transplantation of cells releasing γ-aminobutyric acid (GABA) could be used to counteract the imbalance between excitation and inhibition within epileptic neuronal networks. We generated GABAergic interneuron precursors from human embryonic stem cells (hESCs) and grafted them in the hippocampi of rats developing chronic SRSs after kainic acid-induced status epilepticus. Using whole-cell patch-clamp recordings, we characterised the maturation of the grafted cells into functional GABAergic interneurons in the host brain, and we confirmed the presence of functional inhibitory synaptic connections from grafted cells onto the host neurons. Moreover, optogenetic stimulation of grafted hESC-derived interneurons reduced the rate of epileptiform discharges in vitro. We also observed decreased SRS frequency and total time spent in SRSs in these animals in vivo as compared to non-grafted controls. These data represent a proof-of-concept that hESC-derived GABAergic neurons can exert a therapeutic effect on epileptic animals presumably through establishing inhibitory synapses with host neurons.

CXCL1-CXCR1/2 signaling is induced in human temporal lobe epilepsy and contributes to seizures in a murine model of acquired epilepsy.

CXCL1, a functional murine orthologue of the human chemokine CXCL8 (IL-8), and its CXCR1 and CXCR2 receptors were investigated in a murine model of acquired epilepsy developing following status epilepticus (SE) induced by intra-amygdala kainate. CXCL8 and its receptors were also studied in human temporal lobe epilepsy (TLE). The functional involvement of the chemokine in seizure generation and neuronal cell loss was assessed in mice using reparixin (formerly referred to as repertaxin), a non-competitive allosteric inhibitor of CXCR1/2 receptors. We found a significant increase in hippocampal CXCL1 level within 24 h of SE onset that lasted for at least 1 week. No changes were measured in blood. In analogy with human TLE, immunohistochemistry in epileptic mice showed that CXCL1 and its two receptors were increased in hippocampal neuronal cells. Additional expression of these molecules was found in glia in human TLE. Mice were treated with reparixin or vehicle during SE and for additional 6 days thereafter, using subcutaneous osmotic minipumps. Drug-treated mice showed a faster SE decay, a reduced incidence of acute symptomatic seizures during 48 h post-SE, and a delayed time to spontaneous seizures onset compared to vehicle controls. Upon reparixin discontinuation, mice developed spontaneous seizures similar to vehicle mice, as shown by EEG monitoring at 14 days and 2.5 months post-SE. In the same epileptic mice, reparixin reduced neuronal cell loss in the hippocampus vs vehicle-injected mice, as assessed by Nissl staining at completion of EEG monitoring. Reparixin administration for 2 weeks in mice with established chronic seizures, reduced by 2-fold on average seizure number vs pre-treatment baseline, and this effect was reversible upon drug discontinuation. No significant changes in seizure number were measured in vehicle-injected epileptic mice that were EEG monitored in parallel. Data show that CXCL1-IL-8 signaling is activated in experimental and human epilepsy and contributes to acute and chronic seizures in mice, therefore representing a potential new target to attain anti-ictogenic effects.

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

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

Reactive astrocyte-driven epileptogenesis is induced by microglia initially activated following status epilepticus.

Extensive activation of glial cells during a latent period has been well documented in various animal models of epilepsy. However, it remains unclear whether activated glial cells contribute to epileptogenesis, i.e., the chronically persistent process leading to epilepsy. Particularly, it is not clear whether interglial communication between different types of glial cells contributes to epileptogenesis, because past literature has mainly focused on one type of glial cell. Here, we show that temporally distinct activation profiles of microglia and astrocytes collaboratively contributed to epileptogenesis in a drug-induced status epilepticus model. We found that reactive microglia appeared first, followed by reactive astrocytes and increased susceptibility to seizures. Reactive astrocytes exhibited larger Ca2+ signals mediated by IP3R2, whereas deletion of this type of Ca2+ signaling reduced seizure susceptibility after status epilepticus. Immediate, but not late, pharmacological inhibition of microglial activation prevented subsequent reactive astrocytes, aberrant astrocyte Ca2+ signaling, and the enhanced seizure susceptibility. These findings indicate that the sequential activation of glial cells constituted a cause of epileptogenesis after status epilepticus. Thus, our findings suggest that the therapeutic target to prevent epilepsy after status epilepticus should be shifted from microglia (early phase) to astrocytes (late phase).

Intramuscularly administered A1 adenosine receptor agonists as delayed treatment for organophosphorus nerve agent-induced Status Epilepticus.

Exposure to organophosphorus nerve agents (NAs) like sarin (GB) and soman (GD) can lead to sustained seizure activity, or status epilepticus (SE). Previous research has shown that activation of A1 adenosine receptors (A1ARs) can inhibit neuronal excitability, which could aid in SE termination. Two A1AR agonists, 2-Chloro-N6-cyclopentyladenosine (CCPA) and N-Bicyclo(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA), were effective in terminating GD-induced SE in rats when administered via intraperitoneal (IP) injection. However, IP injection is not a clinically relevant route of administration. This study evaluated the efficacy of these agonists in terminating NA-induced SE when administered via intramuscular (IM) route. Adult male rats were exposed subcutaneously (SC) to either GB (150 µg/kg) or GD (90 µg/kg) and were treated with ENBA or CCPA at 15, 30, or 60 min after seizure onset or left untreated. Up to 7 days after exposure, deeply anesthetized rats were euthanized and perfused brains were removed for histologic assessment of neuropathology (i.e., neuronal damage) in six brain regions (amygdala, cerebral cortex, piriform cortex, thalamus, dorsal hippocampus, and ventral hippocampus). A total neuropathy score (0-24) was determined for each rat by adding the scores from each of the six regions. The higher the total score the more severe the neuropathology. With the GB model and 60 min treatment delay, ENBA-treated rats experienced 78.6% seizure termination (N = 14) and reduced neuropathology (11.6 ± 2.6, N = 5), CCPA-treated rats experienced 85.7% seizure termination (N = 14) and slightly reduced neuropathology (20.7 ± 1.8, N = 6), and untreated rats experienced no seizure termination (N = 13) and severe neuropathology (22.3 ± 1.0, N = 4). With the GD model and 60 min treatment delay, ENBA-treated rats experienced 92.9% seizure termination (N = 14) and reduced neuropathology (13.96 ± 1.8, N = 9), CCPA-treated rats experienced 78.6% seizure termination (N = 14) and slightly reduced neuropathology (22.0 ± 0.9, N = 10); and untreated rats experienced 16.7% seizure termination (N = 12) and severe neuropathology (22.0 ± 1.8, N = 5). While ENBA and CCPA both demonstrate a clear ability to terminate SE when administered up to 60 min after seizure onset, ENBA offers more neuroprotection, making it a promising candidate for NA-induced SE.

Interleukin-1ß-Treated Mesenchymal Stem Cells Inhibit Inflammation in Hippocampal Astrocytes Through Exosome-Activated Nrf-2 Signaling.

BACKGROUND: Interleukin-1ß (IL-1)-treated mesenchymal stem cells (MSCs) and IL-1-MSCs-conditioned medium (CM) exert anti-inflammatory roles. Astrocytes are essential for the modulation of synaptic activity and neuronal homeostasis in the brain. Exosomes are the critical mediators in intercellular communication. However, the mechanism underlying the anti-inflammatory effect of IL-1-treated MSCs remains unknown. METHODS: In this study, exosomes (IL-1-Exo) were isolated from IL-1-treated MSCs. In addition, lipopolysaccharide (LPS)-treated hippocampal astrocytes and status epilepticus (SE) mice were treated with IL-1-Exo. Inflammatory activity, astrogliosis, and cognitive performance were measured to determine the effect of IL-1-Exo on inflammation. RESULTS: The results revealed that IL-1-Exo significantly inhibited LPS-induced astrogliosis and inflammatory responses of astrocytes. Also, IL-1-Exo reversed the LPS-induced effect on calcium signaling. The Nrf2 signaling pathway was associated with the effect of IL-1-Exo in LPS-treated astrocytes. Furthermore, IL-1-Exo reduced the inflammatory response and improved the cognitive performance of SE mice. CONCLUSION: The results suggest that IL-1-Exo inhibited LPS-induced inflammatory responses in astrocytes and SE mice and that the effect of IL-1-Exo was primarily mediated through the Nrf-2 signaling pathway. This study provides a new understanding of the molecular mechanism of inflammation-associated brain diseases and an avenue to develop nanotherapeutic agents for the treatment of inflammatory conditions in the brain.

Changes in the expression of endothelial monocyte­activating polypeptide II in the rat hippocampus following status epilepticus.

Endothelial monocyte­activating polypeptide II (EMAP II) is a sensitive marker of neurotoxic injury, the expression of which increases significantly under conditions of stress, such as hypoxia or apoptosis. Studies have confirmed the extensive apoptosis of nerve cells in the brain following status epilepticus (SE), and the occurrence of SE can confer a hypoxic state on cells. The purpose of the present study was to observe the changes in the expression of EMAP II, and in the numbers and tight junction protein levels of microvascular endothelial cells in the hippocampus of rats with pilocarpine­induced SE. The protein expression levels of EMAP II, CD31, zonula occludens 1 (ZO­1) and occludin in the hippocampus were determined by immunofluorescence and western blot analyses. It was found that almost 75.6% of the rats in the SE group developed Racine stage IV­V seizures at approximately 44.7±18.8 min after the pilocarpine administration, and the 24­h mortality rate was almost 10.4%. The weight of the rats in the SE group was significantly decreased within 24 h following SE. Immunofluorescence staining revealed a low EMAP II expression in the hippocampus of the rats in the control group; however, the numbers of EMAP II­positive cells were significantly increased in the SE group from 2 h to 21 days. The trend of EMAP II protein expression was consistent with that obtained with immunofluorescence staining. The numbers of CD31­positive microvascular endothelial cells were significantly increased from 24 h to 21 days compared with the levels in the control group. The protein expression of ZO­1 and occludin was most significantly decreased in the SE group. On the whole, the present study demonstrated that the expression of EMAP II in the rat hippocampus was upregulated in the SE model, which may promote angiogenesis and alter the TJ integrity of brain microvascular endothelial cells, with an increased number of CD31­positive microvascular endothelial cells and a decreased expression of ZO­1 and occludin.

Hippocampal Sclerosis in Pilocarpine Epilepsy: Survival of Peptide-Containing Neurons and Learning and Memory Disturbances in the Adult NMRI Strain Mouse.

The present experiments reveal the alterations of the hippocampal neuronal populations in chronic epilepsy. The mice were injected with a single dose of pilocarpine. They had status epilepticus and spontaneously recurrent motor seizures. Three months after pilocarpine treatment, the animals were investigated with the Barnes maze to determine their learning and memory capabilities. Their hippocampi were analyzed 2 weeks later (at 3.5 months) with standard immunohistochemical methods and cell counting. Every animal displayed hippocampal sclerosis. The neuronal loss was evaluated with neuronal-N immunostaining, and the activation of the microglia was measured with Iba1 immunohistochemistry. The neuropeptide Y, parvalbumin, and calretinin immunoreactive structures were qualitatively and quantitatively analyzed in the hippocampal formation. The results were compared statistically to the results of the control mice. We detected neuronal loss and strongly activated microglia populations. Neuropeptide Y was significantly upregulated in the sprouting axons. The number of parvalbumin- and calretinin-containing interneurons decreased significantly in the Ammon's horn and dentate gyrus. The epileptic animals displayed significantly worse learning and memory functions. We concluded that degeneration of the principal neurons, a numerical decrease of PV-containing GABAergic neurons, and strong peptidergic axonal sprouting were responsible for the loss of the hippocampal learning and memory functions.

Dynamic alteration of dendrites and dendritic spines in the hippocampus and microglia in mouse brain tissues after kainate-induced status epilepticus.

PURPOSE: To study the alteration of microglial subtypes, the representative markers of microglia, and the morphology of dendrites and dendritic spines after acute status epilepticus (SE) and during recurrent seizures. METHODS: A mouse kainate-induced SE model was used. Dendrites and dendritic spines of granule neurons in the dentate gyrus (DG) subregion and pyramidal neurons in the cornu ammonis (CA)1 and cornu ammonis (CA)3 subregions of the hippocampus were visualized by Golgi staining. Synaptic proteins were evaluated by Western blot analysis, and microglia and their markers were evaluated by flow cytometry. RESULTS: Extensive partial spine loss was observed in the dendrites of granule and pyramidal cells in the acute and early chronic stages of SE. In terms of spine loss, the thin and mushroom types predominated. Accompanying the spine loss in these two stages, the proportion of M1 microglia increased significantly with high CX3CR1 expression and low CD200R expression. However, at the transiting stage, the proportion of M2 microglia was increased dramatically, and high expression levels of CXCR3 on all microglia and CD68 on M1 microglia were observed. Morris water maze tests revealed significant learning and memory impairment in the chronic phase of epilepsy. CONCLUSION: Dendritic spines in the hippocampus and microglia in the central nevus system are dynamically altered in epilepsy during the establishment and maintenance of spontaneous seizures. Microglia may contribute to the spine loss and related learning and memory impairment.

Peroxisome Proliferator-Activated Receptor γ Coactivator 1α Activates Vascular Endothelial Growth Factor That Protects Against Neuronal Cell Death Following Status Epilepticus through PI3K/AKT and MEK/ERK Signaling.

Status epilepticus may cause molecular and cellular events, leading to hippocampal neuronal cell death. Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) is an important regulator of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2), also known as fetal liver kinase receptor 1 (Flk-1). Resveratrol is an activator of PGC-1α. It has been suggested to provide neuroprotective effects in epilepsy, stroke, and neurodegenerative diseases. In the present study, we used microinjection of kainic acid into the left hippocampal CA3 region in Sprague Dawley rats to induce bilateral prolonged seizure activity. Upregulating the PGC-1α pathway will increase VEGF/VEGFR2 (Flk-1) signaling and further activate some survival signaling that includes the mitogen activated protein kinase kinase (MEK)/mitogen activated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways and offer neuroprotection as a consequence of apoptosis in the hippocampal neurons following status epilepticus. Otherwise, downregulation of PGC-1α by siRNA against pgc-1α will inhibit VEGF/VEGFR2 (Flk-1) signaling and suppress pro-survival PI3K/AKT and MEK/ERK pathways that are also accompanied by hippocampal CA3 neuronal cell apoptosis. These results may indicate that the PGC-1α induced VEGF/VEGFR2 pathway may trigger the neuronal survival signaling, and the PI3K/AKT and MEK/ERK signaling pathways. Thus, the axis of PGC-1α/VEGF/VEGFR2 (Flk-1) and the triggering of downstream PI3K/AKT and MEK/ERK signaling could be considered an endogenous neuroprotective effect against apoptosis in the hippocampus following status epilepticus.

Promising Opportunities for Treating Neurodegenerative Diseases with Mesenchymal Stem Cell-Derived Exosomes.

Neurodegenerative disease refers to any pathological condition in which there is a progressive decline in neuronal function resulting from brain atrophy. Despite the immense efforts invested over recent decades in developing treatments for neurodegenerative diseases, effective therapy for these conditions is still an unmet need. One of the promising options for promoting brain recovery and regeneration is mesenchymal stem cell (MSC) transplantation. The therapeutic effect of MSCs is thought to be mediated by their secretome, and specifically, by their exosomes. Research shows that MSC-derived exosomes retain some of the characteristics of their parent MSCs, such as immune system modulation, regulation of neurite outgrowth, promotion of angiogenesis, and the ability to repair damaged tissue. Here, we summarize the functional outcomes observed in animal models of neurodegenerative diseases following MSC-derived exosome treatment. We will examine the proposed mechanisms of action through which MSC-derived exosomes mediate their therapeutic effects and review advanced studies that attempt to enhance the improvement achieved using MSC-derived exosome treatment, with a view towards future clinical use.