Pentylenetetrazole kindling induces dynamic changes in GAD65 expression in hippocampal somatostatin interneurons.

INTRODUCTION: One of the mechanisms of epileptgenesis is impairment of inhibitory neural circuits. Several studies have compared neural changes among subtypes of gamma-aminobutyric acid-related (GABAergic) neurons after acquired epileptic seizure. However, it is unclear that GABAergic neural modifications that occur during acquisition process of epileptic seizure. METHODS: Male rats were injected with pentylenetetrazole (PTZ kindling: n = 30) or saline (control: n = 15) every other day to observe the development of epileptic seizure stages. Two time points were identified: the point at which seizures were most difficult to induce, and the point at which seizures were most easy to induce. The expression of GABAergic neuron-related proteins in the hippocampus was immunohistochemically compared among GABAergic subtypes at each of these time points. RESULTS: Bimodal changes in seizure stages were observed in response to PTZ kindling. The increase of seizure stage was transiently suppressed after 8 or 10 injections, and then progressed again by the 16th injection. Based on these results, we defined 10 injections as a short-term injection period during which seizures are less likely to occur, and 20 injections as a long-term injection period during which continuous seizures are likely to occur. The immunohistochemical analysis showed that hippocampal glutamic acid decarboxylase 65 (GAD65) expression was increased after short-term kindling but unchanged after long-term kindling. Increased GAD65 expression was limited to somatostatin-positive (SOM+) cells among several GABAergic subtypes. By contrast, GAD, GABA, GABAAR α1, GABABR1, and VGAT cells showed no change following short- or long-term PTZ kindling. CONCLUSION: PTZ kindling induces bimodal changes in the epileptic seizure stage. Seizure stage is transiently suppressed after short-term PTZ injection with GAD65 upregulation in SOM+ cells. The seizure stage is progressed again after long-term PTZ injection with GAD65 reduction to baseline level.

The effect of valproate on the amino acids, monoamines, and kynurenic acid concentrations in brain structures involved in epileptogenesis in the pentylenetetrazol-kindled rats.

BACKGROUND: The study aimed to assess the influence of a single valproate (VPA) administration on inhibitory and excitatory neurotransmitter concentrations in the brain structures involved in epileptogenesis in pentylenetetrazol (PTZ)-kindled rats. METHODS: Adult, male Wistar rats were kindled by repeated intraperitoneal (ip) injections of PTZ at a subconvulsive dose (30 mg/kg, three times a week). Due to the different times required to kindle the rats (18-22 injections of PTZ), a booster dose of PTZ was administrated 7 days after the last rats were kindled. Then rats were divided into two groups: acute administration of VPA (400 mg/kg) or saline given ip. The concentration of amino acids, kynurenic acid (KYNA), monoamines, and their metabolites in the prefrontal cortex, hippocampus, amygdala, and striatum was assessed by high-pressure liquid chromatography (HPLC). RESULTS: It was found that a single administration of VPA increased the gamma-aminobutyric acid (GABA), tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), and KYNA concentrations and decreased aspartate (ASP) levels in PTZ-kindled rats in the prefrontal cortex, hippocampus, amygdala and striatum. CONCLUSIONS: Our results indicate that a single administration of VPA in the PTZ-kindled rats restored proper balance between excitatory (decreasing the level of ASP) and inhibitory neurotransmission (increased concentration GABA, KYNA) and affecting serotoninergic neurotransmission in the prefrontal cortex, hippocampus, amygdala, and striatum.

Hippocampal tandem mass tag (TMT) proteomics analysis during kindling epileptogenesis in rat.

Epilepsy is a neurological disorder that remains difficult to treat due to the lack of a clear molecular mechanism and incomplete understanding of involved proteins. To identify potential therapeutic targets, it is important to gain insight into changes in protein expression patterns related to epileptogenesis. One promising approach is to analyze proteomic data, which can provide valuable information about these changes. In this study, to evaluate the changes in gene expression during epileptogenesis, LC-MC2 analysis was carried out on hippocampus during stages of electrical kindling in rat models. Subsequently, progressive changes in the expression of proteins were detected as a result of epileptogenesis development. In line with behavioral kindled seizure stages and according to the proteomics data, we described epileptogenesis phases by comparing Stage3 versus Control (S3/C0), Stage5 versus Stage3 (S5/S3), and Stage5 versus Control group (S5/C0). Gene ontology analysis on differentially expressed proteins (DEPs) showed significant changes of proteins involved in immune responses like Csf1R, Aif1 and Stat1 during S3/C0, regulation of synaptic plasticity like Bdnf, Rac1, CaMK, Cdc42 and P38 during S5/S3, and nervous system development throughout S5/C0 like Bdnd, Kcc2 and Slc1a3.There were also proteins like Cox2, which were altered commonly among all three phases. The pathway enrichment analysis of DEPs was also done to discover molecular connections between phases and we have found that the targets like Csf1R, Bdnf and Cox2 were analyzed throughout all three phases were highly involved in the PPI network analysis as hub nodes. Additionally, these same targets underwent changes which were confirmed through Western blotting. Our results have identified proteomic patterns that could shed light on the molecular mechanisms underlying epileptogenesis which may allow for novel targeted therapeutic strategies.

Enhanced Astrocyte Activity and Excitatory Synaptic Function in the Hippocampus of Pentylenetetrazole Kindling Model of Epilepsy.

Epilepsy is a chronic condition characterized by recurrent spontaneous seizures. The interaction between astrocytes and neurons has been suggested to play a role in the abnormal neuronal activity observed in epilepsy. However, the exact way astrocytes influence neuronal activity in the epileptogenic brain remains unclear. Here, using the PTZ-induced kindling mouse model, we evaluated the interaction between astrocyte and synaptic function by measuring astrocytic Ca2+ activity, neuronal excitability, and the excitatory/inhibitory balance in the hippocampus. Compared to control mice, hippocampal slices from PTZ-kindled mice displayed an increase in glial fibrillary acidic protein (GFAP) levels and an abnormal pattern of intracellular Ca2+-oscillations, characterized by an increased frequency of prolonged spontaneous transients. PTZ-kindled hippocampal slices also showed an increase in the E/I ratio towards excitation, likely resulting from an augmented release probability of excitatory inputs without affecting inhibitory synapses. Notably, the alterations in the release probability seen in PTZ-kindled slices can be recovered by reducing astrocyte hyperactivity with the reversible toxin fluorocitrate. This suggests that astroglial hyper-reactivity enhances excitatory synaptic transmission, thereby impacting the E/I balance in the hippocampus. Altogether, our findings support the notion that abnormal astrocyte-neuron interactions are pivotal mechanisms in epileptogenesis.

Silencing of dentate gyrus inhibits mossy fiber sprouting and prevents epileptogenesis through NDR2 kinase in pentylenetetrazole kindling rat model of TLE.

Epileptogenesis is a potential process. Mossy fiber sprouting (MFS) contributes to epileptogenesis. Silencing of the dentate gyrus (DG) suppressed spontaneous seizures model of epilepsy and hyperactivity of granule cells resulted in MFS in vitro. However, the role of DG's excitability in epileptogenesis have not yet been well explored, and underlying mechanisms has not been elucidated. Using chemical genetics, we studied whether MFS and epileptogenesis could be modulated by silencing of DG in the PTZ kindling rat model of epilepsy. MFS and protein expression was measured by Timm staining, Western blotting, and Immunofluorescence. Previous studies demonstrated that MFS and epileptogenesis could be modulated by a regulator of axonal growth (e.g. RGMa, PTEN). NDR2 kinase regulate neuronal polarity and prevents the formation of supernumerary axons in the hippocampus. We experimentally confirmed chemogenetic inhibition in DG resulted in decreased MFS and NDR2 expression, and alleviated epileptogenesis. Furthermore, our results showed that injection of AVV vector expressing NDR2 into DG induced upregulation of NDR2 in the hippocampus, and over expression of NDR2 in the hippocampus promote MFS and block protective effect of chemogenetic silencing of DG on epileptogenesis. Overall, we concluded that silencing of DG inhibits MFS and prevents epileptogenesis through NDR2 in the hippocampus in the PTZ kindling rat model of TLE, thereby providing a possible strategy to prevent epileptogenesis.

iTRAQ-based proteomic analysis of the hippocampus of pentylenetetrazole-kindled epileptic rats.

Epilepsy can severely affect the quality of life of patients, who are often at higher risk of mortality. However, the molecular mechanisms and pathogenesis underlying epileptogenesis are poorly understood. In this study, we performed a proteomic analysis of the hippocampus in pentylenetetrazole (PTZ)-kindled epileptic rats to explore the molecular mechanisms of epileptogenesis. We established an epileptic model in Sprague Dawley rats by injecting PTZ intraperitoneally and applied isobaric tags for relative and absolute quantification (iTRAQ) technology integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins (DEPs) in the hippocampus. A total of 99 proteins, comprising 93 upregulated and 6 downregulated proteins, were identified based on a fold change >1.2 (or <0.83) and a p-value < .05. A further bioinformatics analysis suggested that the candidate proteins were mainly involved in the ubiquitin ligase complex or metabolite homeostasis or acted as intrinsic components of the membrane. A Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment analysis identified a series of representative pathological pathways, including the calcium signaling pathway, neuroactive ligand-receptor interaction pathway, and the NF-kappa B pathway. The mass spectrometry results were further confirmed by assessing five representative proteins (Akt1, Syvn1, Amfr, Lamb1, and Cox17) using western blotting and immunohistochemistry. These results may help to reveal the molecular mechanisms underlying epileptogenesis and provide new directions or targets for epilepsy research.

Ultrastructural GABA immunogold labeling in the substantia nigra pars reticulata of kindled genetic absence epilepsy rats.

Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is a well-known animal model of absence epilepsy and they are resistant to electrical kindling stimulations. The present study aimed to examine possible differences in gamma-aminobutyric acid (GABA) levels and synapse counts in the substantia nigra pars reticulata anterior (SNRa) and posterior (SNRp) regions between GAERS and Wistar rats receiving kindling stimulations. Animals in the kindling group either received six stimulations in the amygdala and had grade 2 seizures or they were kindled, having grade five seizures. Rats were decapitated one hour after the last stimulation. SNR regions were obtained after vibratome sectioning of the brain tissue. GABA immunoreactivity was detected by immunogold method and synapses were counted. Sections were observed by transmission electron microscope and analyzed by Image J program. GABA density in the SNRa region of fully kindled GAERS and Wistar groups increased significantly compared to that of their corresponding grade 2 groups. The number of synapses increased significantly in kindled and grade 2 GAERS groups, compared to kindled and grade 2 Wistar groups, respectively, in the SNRa region. GABA density in the SNRp region of kindled GAERS group increased significantly compared to that of GAERS grade 2 group. In the SNRp region, both kindled and grade 2 GAERS groups were found to have increased number of synapses compared to that of GAERS control group. We concluded that both SNRa and SNRp regions may be important in modulating resistance of GAERS to kindling stimulations.

Characterization of kindled VGAT-Cre mice as a new animal model of temporal lobe epilepsy.

OBJECTIVE: Development of novel therapies for temporal lobe epilepsy is hindered by a lack of models suitable for drug screening. While testing the hypothesis that "inhibiting inhibitory neurons" was sufficient to induce seizures, it was discovered that a mild electrical kindling protocol of VGAT-Cre mice led to spontaneous motor and electrographic seizures. This study characterizes these seizures and investigates the mechanism. METHODS: Mice were implanted with electroencephalographic (EEG) headsets that included a stimulating electrode in the hippocampus before being electrically kindled. Seizures were evaluated by review of EEG recordings and behavior. γ-Aminobutyric acidergic (GABAergic) neurotransmission was evaluated by quantitative polymerase chain reaction, immunocytochemistry, Western blot, and electrophysiology. RESULTS: Electrical kindling of VGAT-Cre mice induces spontaneous recurring seizures after a short latency (6 days). Seizures occur 1-2 times per day in both male and female mice, with only minimal neuronal death. These mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT), a gene that is specifically expressed in GABAergic inhibitory neurons. The insertion of Cre disrupts the expression of VGAT mRNA and protein, and impairs GABAergic synaptic transmission in the hippocampus. SIGNIFICANCE: Kindled VGAT-Cre mice can be used to study the mechanisms involved in epileptogenesis and may be useful for screening novel therapeutics.

Pentylenetetrazol kindling induces cortical astrocytosis and increased expression of extracellular matrix molecules in mice.

Although epilepsy is one of the most common chronic neurological disorders with a prevalence of approximately 1.0 %, the underlying pathophysiology remains to be elucidated. Understanding the molecular and cellular mechanisms involved in the development of epilepsy is important for the development of appropriate therapeutic strategy. In this study, we investigated the effects of status epilepticus on astrocytes, microglia, and extracellular matrix (ECM) molecules in the somatosensory cortex and piriform cortex of mice. Activation of astrocytes was observed in many cortices except the retrosplenial granular cortex after pentylenetetrazol (PTZ)-induced kindling in mice. Activated astrocytes in the cortex were found in layers 1-3 but not in layers 4-6. In the somatosensory and piriform cortices, no change was observed in the number of parvalbumin (PV)-positive neurons and PV-positive neurons covered with perineuronal nets. However, the amount of ECM in the extracellular space increased. The expression of VGLUT1- and GAD67-positive synapses also increased. Thus, in the PTZ-kindling epilepsy mice model, an increase in the number of ECM molecules and activation of astrocytes were observed in the somatosensory cortex and piriform cortex. These results indicate that PTZ-induced seizures affect not only the hippocampus but also other cortical areas. Our study findings may help to develop new therapeutic approaches to prevent seizures or their sequelae.

Dynamic oxygen changes during status epilepticus and subsequent endogenous kindling.

OBJECTIVE: Brain tissue oxygen (partial oxygen pressure [pO2 ]) levels are tightly regulated to stay within the normoxic zone, with deviations on either side resulting in impaired brain function. Whereas pathological events such as ischemic attacks and brief seizures have previously been shown to result in pO2 levels well below the normoxic zone, oxygen levels during prolonged status epilepticus (SE) and the subsequent endogenous kindling period are unknown. METHODS: We utilized two models of acquired temporal lobe epilepsy in rats: intrahippocampal kainic acid infusion and prolonged perforant pathway stimulation. Local tissue oxygen was measured in the dorsal hippocampus using an optode during and for several weeks following SE. RESULTS: We observed hyperoxia in the hippocampus during induced SE in both models. Following termination of SE, 88% of rats initiated focal self-generated spiking activity in the hippocampus within the first 7 days, which was associated with dynamic oxygen changes. Self-generated and recurring epileptiform activity subsequently organized into higher-frequency bursts that became progressively longer and were ultimately associated with behavioral seizures that became more severe with time and led to postictal hypoxia. SIGNIFICANCE: Induced SE and self-generated recurrent epileptiform activity can have profound and opposing effects on brain tissue oxygenation that may serve as a biomarker for ongoing pathological activity in the brain.

Inflammatory markers in the hippocampus after audiogenic kindling.

Here, we investigated the participation of pro and anti-inflammatory cytokines in the spread of repeated audiogenic seizures from brainstem auditory structures to limbic areas, including the hippocampus. We used Wistar Audiogenic Rats (WARs) and Wistars submitted to the audiogenic kindling protocol with a loud broad-band noise. We measured pro and anti-inflammatory cytokines and nitrate levels in the hippocampus of stimulated animals. Our results show that all WARs developed audiogenic seizures that evolved to limbic seizures whereas seizure-resistant controls did not present any seizures. However, regardless of seizure severity, we did not observe differences in the pro inflammatory cytokines IL-1ß, IL-6, TNF-α and IFN-α or in the anti-inflammatory IL-10 in the hippocampi of audiogenic and resistant animals. We also did not find any differences in nitrate content. Our data indicate that the spread of seizures during the audiogenic kindling is not dependent on hippocampal release of cytokines or oxidative stress, but the severity of brainstem seizures will be higher in animals with higher levels of cytokines and the oxidative stress marker, nitrate.

Loss of presenilin 2 age-dependently alters susceptibility to acute seizures and kindling acquisition.

Patients with Alzheimer's disease (AD) experience seizures at higher rates than the general population of that age, suggesting an underexplored role of hyperexcitability in AD. Genetic variants in presenilin (PSEN) 1 and 2 genes lead to autosomal dominant early-onset AD (ADAD); patients with PSEN gene variants also report seizures. Pharmacological control of seizures in AD may be disease-modifying. Preclinical efficacy of FDA-approved antiseizure drugs (ASDs) is well defined in young adult rodents; however, the efficacy of ASDs in aged rodents with chronic seizures is less clear. The mechanism by which ADAD genes lead to AD remains unclear, and even less studied is the pathogenesis of epilepsy in AD. PSEN variants generally all result in a biochemical loss of function (De Strooper, 2007). We herein determined whether well-established models of acute and chronic seizure could be used to explore the relationship between AD genes and seizures through investigating whether loss of normal PSEN2 function age-dependently influenced susceptibility to seizures and/or corneal kindling acquisition. PSEN2 knockout (KO) and age-matched wild-type (WT) mice were screened from 2- to 10-months-old to establish age-dependent focal seizure threshold. Additionally, PSEN2 KO and WT mice aged 2- and 8-months-old underwent corneal kindling such that mice were aged 3- and 9-months old at the beginning of ASD efficacy testing. We then defined the dose-dependent efficacy of mechanistically distinct ASDs on kindled seizures of young versus aged mice to better understand the applicability of corneal kindling to real-world use for geriatric patients. PSEN2 KO mice demonstrated early-life reductions in seizure threshold. However, kindling acquisition was delayed in 2-month-old PSEN2 KO versus WT mice. Young male WT mice took 24.3 ± 1.3 (S.E.M.) stimulations to achieve kindling criterion, whereas age-matched PSEN2 KO male mice took 41.2 ± 1.1 stimulations (p < .0001). The rate of kindling acquisition of 8-month-old mice was no longer different from WT. This study demonstrates that loss of normal PSEN2 function is associated with age-dependent changes in the in vivo susceptibility to acute seizures and kindling. Loss of normal PSEN2 function may be an underexplored molecular contributor to seizures. The use of validated models of chronic seizures in aged rodents may uncover age-related changes in susceptibility to epileptogenesis and/or ASD efficacy in mice with AD-associated genotypes, which may benefit the management of seizures in AD.

Activation of adenosine receptors modulates the efflux transporters in brain capillaries and restores the anticonvulsant effect of carbamazepine in carbamazepine resistant rats developed by window-pentylenetetrazole kindling.

Up-regulation of efflux transporters in brain capillaries may lead to the decreased therapeutic efficacy of antiepileptic drugs in patients with Drug Resistant Epilepsy. Adenosine receptor activation in brain capillaries can modulate blood-brain barrier permeability by decreasing the protein levels and function of efflux transporters. Therefore, we aimed to investigate whether the activation of adenosine receptors improves convulsions outcome in carbamazepine (CBZ) resistant animals and modulates the protein levels of efflux transporters (P-GP, MRP1, MRP2) in brain capillaries. We employed the window-pentylenetetrazol (PTZ) kindling model to develop CBZ resistant rats by CBZ administration during the post-kindling phase, and tested if these animals displayed subsequent resistance to other antiepileptic drugs. Crucially, we investigated if the administration of a broad-spectrum adenosine agonist (NECA) improves convulsions control in CBZ resistant rats. Of potential therapeutic relevance, in CBZ resistant rats NECA restored the anticonvulsant effect of CBZ. We also evaluated how the resistance to CBZ and the activation of adenosine receptors with NECA affect protein levels of efflux transporters in brain capillaries, as quantified by western blot. While CBZ resistance was associated with the up-regulation of both P-GP/MRP2 in brain capillaries, with the administration of NECA in CBZ resistant rats, we observed a decrease of P-GP and an increase of MRP2 levels, in brain capillaries. Since the activation of adenosine receptors improves the outcome of convulsions probably through the modulation of the efflux transporters protein levels in brain capillaries, adenosine agonists could be useful as an adjunct therapy for the control of Drug Resistant Epilepsy.

Spatial exploration induced expression of immediate early genes Fos and Zif268 in adult-born neurons Is reduced after pentylenetetrazole kindling.

Seizure activity stimulates adult neurogenesis, the birth of new neurons, in the hippocampus. Many new neurons that develop in the presence of repeatedly induced seizures acquire abnormal morphological and functional characteristics that can promote network hyperexcitability and hippocampal dysfunction. However, the impact of seizure induced neurogenesis on behaviour remains poorly understood. In this study, we investigated whether adult-born neurons generated immediately before and during chronic seizures were capable of integration into behaviorally relevant hippocampal networks. Adult rats underwent pentylenetetrazole (PTZ) kindling for either 1 or 2 weeks. Proliferating cells were labelled with BrdU immediately before kindling commenced. Twenty-four hours after receiving their last kindling treatment, rats were placed in a novel environment and allowed to freely explore for 30 min. The rats were euthanized 90 min later to examine for behaviourally-induced immediate early gene expression (c-fos, Zif268). Using this approach, we found that PTZ kindled rats did not differ from control rats in regards to exploratory behaviour, but there was a marked attenuation in behaviour-induced expression of Fos and Zif268 for rats that received 2 weeks of PTZ kindling. Further examination revealed that PTZ kindled rats showed reduced colocalization of Fos and Zif268 in 2.5 week old BrdU + cells. The proportion of immature granule cells (doublecortin-positive) expressing behaviorally induced Zif268 was also significantly lower for PTZ kindled rats than control rats. These results suggest that chronic seizures can potentially disrupt the ability of adult-born cells to functionally integrate into hippocampal circuits important for the processing of spatial information.

PET Neuroimaging Reveals Serotonergic and Metabolic Dysfunctions in the Hippocampal Electrical Kindling Model of Epileptogenesis.

Glucose metabolism and serotonergic neurotransmission have been reported to play an important role in epileptogenesis. We therefore aimed to use neuroimaging to evaluate potential alterations in serotonin 5-HT1A receptor and glucose metabolism during epileptogenesis in the rat electrical kindling model. To achieve this goal, we performed positron emission tomography (PET) imaging in a rat epileptogenesis model triggered by electrical stimulation of the hippocampus using 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG), a radiolabeled analog of glucose, and 2'-methoxyphenyl-(N-2'-pyridinyl)-p-18F-fluoro-benzamidoethylpiperazine (18F-MPPF), a radiolabeled 5-HT1A receptor ligand, to evaluate brain metabolism and 5-HT1A receptor functionality. Since the 5-HT1A receptor is also highly expressed in astrocytes, glial fibrillary acidic protein (GFAP) immunofluorescence was performed to detect astrogliosis arising from the kindling procedure once the study was finalized. Lastly, in vitro18F-MPPF autoradiography was performed to evaluate changes in 5HT1A receptor expression. 18F-FDG PET showed reduction of glucose uptake in cortical structures, whereas 18F-MPPF PET revealed an enhancement of tracer binding potential (BPND) in key areas rich in 5-HT1A receptor involved in epilepsy, including septum, hippocampus and entorhinal cortex of kindled animals compared to controls. However, in vitro 5-HT1A receptor autoradiography showed no changes in densitometric signal in any brain region, suggesting that the augmentation in BPND found by PET could be caused by reduction of synaptic serotonin. Importantly, astroglial activation was detected in the hippocampus of kindled rats. Overall, electrical kindling induced hypometabolism, astrogliosis and serotonergic alterations in epilepsy-related regions. Furthermore, the present findings point to 5-HT1A receptor as a valuable epileptogenesis biomarker candidate and a potential therapeutic target.

The role of UCH-L1, MMP-9, and GFAP as peripheral markers of different susceptibility to seizure development in a preclinical model of epilepsy.

In our study, we assessed the potency of the brain-derived proteins ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), matrix metalloproteinase 9 (MMP-9), glial fibrillary acidic protein (GFAP) and the immune activation indicators interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) as peripheral biomarkers of different susceptibilities to kindling in a preclinical model. We observed increased plasma UCH-L1 levels in kindled vs. control animals. Furthermore, MMP-9 and IL-1ß concentrations were the lowest in rats resistant to kindling. In summary, UCH-L1 is an indicator of neuronal loss and BBB disruption after seizure. MMP-9 and IL-1ß may indicate resistance to kindling. UCH-L1, MMP-9 and IL-1ß may have utility as peripheral biomarkers with translational potency in the clinic.

P53 knockout mice are protected from cocaine-induced kindling behaviors via inhibiting mitochondrial oxidative burdens, mitochondrial dysfunction, and proapoptotic changes.

Previously we demonstrated that p53 mediates dopaminergic neurotoxicity via inducing mitochondrial burdens and proapoptotsis. However, little is known about the role of p53 in the excitotoxicity induced by psychostimulant, such as cocaine. Cocaine-induced kindling (convulsive) behaviors significantly increased p53 expression in the brain. Cocaine-induced p53 expression was more pronounced in hippocampus than in striatum or prefrontal cortex. Genetic depletion of p53 significantly attenuated cocaine-induced convulsive behaviors, followed by c-Fos immunoreactivity, and oxidative burdens in the hippocampus of mice. The antioxidant potentials mediated by genetic depletion of p53 were more pronounced in the mitochondrial-than cytosolic-fraction. Depletion of p53 significantly attenuated the changes in mitochondrial transmembrane potential, intramitochondrial Ca2+ level, and mitochondrial oxidative burdens induced by cocaine. Consistently, depletion of p53 significantly inhibited mitochondrial p53 translocation, and cleaved-PKCδ induced by cocaine. In addition, depletion of p53 protected from cytosolic cytochrome c release, and pro-apoptotic changes induced by cocaine. Importantly, the protective/anticonvulsant potentials by genetic depletion of p53 were comparable to those by pifithrin-µ (PFT), a p53 inhibitor. Our results suggest that depletion of p53 offers anticonvulsive and neuroprotective potentials mainly via attenuating mitochondrial oxidative burdens, mitochondrial dysfunction, and pro-apoptotic signalings against cocaine-induced convulsive neurotoxicity.

Up-regulation of antioxidative proteins TRX1, TXNL1 and TXNRD1 in the cortex of PTZ kindling seizure model mice.

Oxidative stress has been considered as one of pathogenesis of brain damage led by epilepsy. Reducing oxidative stress can ameliorate brain damage during seizures. However, expression levels of important antioxidative enzymes such as thioredoxin-1 (TRX1), thioredoxin-like 1 protein (TXNL1) and thioredoxin reductase 1 (TXNRD1) during seizures have not been investigated. In this study, we examined protein and mRNA expression levels of TRX1, TXNL1 and TXNRD1 in different brain regions in PTZ induced seizure model mice. We found that protein expression levels of TRX1, TXNL1 and TXNRD1 are simultaneously up-regulated by 2- or 3-fold in the cortex of both acute and chronic seizure model mice. But there is no unified expression pattern change of these enzymes in the hippocampus, cerebellum and diencephalon in the seizure model mice. Less extent up-regulation of mRNA expression of these enzymes were also observed in the cortex of seizure mice. These data suggest that antioxidative enzymes may provide a protective effect against oxidative stress in the cortex during seizures.

Astrocytic mobilization of glutathione peroxidase-1 contributes to the protective potential against cocaine kindling behaviors in mice via activation of JAK2/STAT3 signaling.

Compelling evidence indicates that oxidative stress contributes to cocaine neurotoxicity. The present study was performed to elucidate the role of the glutathione peroxidase-1 (GPx-1) in cocaine-induced kindling (convulsive) behaviors in mice. Cocaine-induced convulsive behaviors significantly increased GPx-1, p-IkB, and p-JAK2/STAT3 expression, and oxidative burdens in the hippocampus of mice. There was no significant difference in cocaine-induced p-IkB expression between non-transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) mice, but significant differences were observed in cocaine-induced p-JAK2/STAT3 expression and oxidative stress between non-TG and GPx-1 TG mice. Cocaine-induced glial fibrillary acidic protein (GFAP)-labeled astrocytic level was significantly higher in the hippocampus of GPx-1 TG mice. Triple-labeling immunocytochemistry indicated that GPx-1-, p-STAT3-, and GFAP-immunoreactivities were co-localized in the same cells. AG490, a JAK2/STAT3 inhibitor, but not pyrrolidone dithiocarbamate, an NFκB inhibitor, significantly counteracted GPx-1-mediated protective potentials (i.e., anticonvulsant-, antioxidant-, antiapoptotic-effects). Genetic overexpression of GPx-1 significantly attenuated proliferation of Iba-1-labeled microglia induced by cocaine in mice. However, AG490 or astrocytic inhibition (by GFAP antisense oligonucleotide and α-aminoadipate) significantly increased Iba-1-labeled microglial activity and M1 phenotype microglial mRNA levels, reflecting that proinflammatory potentials were mediated by AG490 or astrocytic inhibition. This microglial activation was less pronounced in GPx-1 TG than in non-TG mice. Furthermore, either AG490 or astrocytic inhibition significantly counteracted GPx-1-mediated protective potentials. Therefore, our results suggest that astrocytic modulation between GPx-1 and JAK2/STAT3 might be one of the underlying mechanisms for protecting against convulsive neurotoxicity induced by cocaine.

Genetic and Pharmacological Targeting of Heat Shock Protein 70 in the Mouse Amygdala-Kindling Model.

Inflammatory responses involving Toll-like receptor signaling represent a key factor contributing to epileptogenesis. Thus, it is of particular interest to explore the relevance of toll-like receptor ligands and modulators, such as heat shock protein 70 (HSP70). Motivated by recent findings demonstrating an upregulation of HSP70 in a model of epileptogenesis, we analyzed the consequences of genetic and pharmacological targeting of HSP70 expression in a mouse kindling paradigm. Lack of inducible HSP70 resulted in increased prekindling seizure thresholds. However, at threshold stimulation the deficiency-promoted seizure spread, as indicated by an increased seizure severity. Subsequent kindling stimulations elicited more severe seizures in knockout mice, whereas endogenous termination of seizure activity remained unaffected with duration of behavioral and electrographic seizure activity comparable to that of wild-type mice. Interestingly, HSP70 deficiency resulted in enhanced microglia activation in the CA1 region. Next, we assessed a pharmacological targeting approach aiming to promote HSP70 expression. Celastrol treatment had no impact on kindling progression but reduced postkindling seizure thresholds and enhanced microglia activation in CA1 and CA3. In conclusion, the findings from HSP70-knockout mice support a protective role of HSP70 with an effect on microglia activation and spread of seizure activity. Unexpectedly, celastrol administration resulted in detrimental consequences. These findings should be considered as a warning about the general safety of celastrol as a drug candidate. The impact of pathophysiological mechanisms on the quality of celastrol effects requires comprehensive future studies exploring influencing factors. Moreover, alternate strategies to increase HSP70 expression should be further developed and validated.