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.

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.

Non-invasive mapping of glutathione levels in mouse brains by in vivo electron paramagnetic resonance (EPR) imaging: Applied to a kindling mouse model.

Glutathione (GSH) is an important antioxidant that can protect cells under oxidative stress. Thus, a non-invasive method to measure and map the distribution of GSH in live animals is needed. To image the distribution of GSH levels in specific brain regions, a new method using electron paramagnetic resonance (EPR) imaging with a nitroxide imaging probe was developed. Pixel-based mapping of brain GSH levels was successfully obtained by using the linear relationship between reduction rates for nitroxides in brains, measured by an in vivo EPR imager, and brain GSH levels, measured by an in vitro biochemical assay. The newly developed method was applied to a kindling mouse model induced with pentylenetetrazole (PTZ) to visualize changes in GSH levels in specific brain regions after seizure. The obtained map of brain GSH levels clearly indicated decreased GSH levels around the hippocampal region compared to control mice.

Increase Signaling of Wnt/ß-Catenin Pathway and Presence of Apoptosis in Cerebellum of Kindled Rats.

BACKGROUND: Epilepsy is one of the most common neurological disorders in humans, and the role of the cerebellum in its physiopathology remains the subject of study. The Purkinje cells (PC), whose axons target the dentate and interpositus nuclei, form the main cerebellar output to forebrain structures involved in epilepsy. Cerebellar atrophy related to loss of PC has been reported in chronic epilepsy although its mechanism remains unclear. Taking into account that an overexpression of ß-Catenin has been related with cell death, here we present the signaling of ß-Catenin and the type of PC death in cerebellum of rats with seizures induced by the amygdaloid kindling model. METHOD: Using an immunohistochemistry and western blot assay for ß-Catenin, c-Myc, cyclin D3, TUNEL and caspase-3, in rats chronically implanted with electrodes, receiving 0, 3, 15, and 45 electrical stimuli. RESULTS: We found that such rats suffering a major number of stimuli showed the highest amount of marks assessed. CONCLUSION: We concluded that there is a higher activity of the Wnt/ß-Catenin pathway associated with increased number of stimuli may be related with the presence of apoptosis in the cerebellum treated with amygdala kindling. In this way, we suggest this pathway as one of the mechanisms by which cerebellar neurons death in generalized seizures.

Epileptogenic effects of G protein-coupled estrogen receptor 1 in the rat pentylenetetrazole kindling model of epilepsy.

BACKGROUND: G protein-coupled estrogen receptor 1 (GPER-1) has been demonstrated in several parts of the brain and may play an important role in estrogen downstream signaling pathway. However, the effects of this receptor on epileptic seizure are not clearly known. Therefore, the effects of GPER-1 agonist, G-1, GPER-1 antagonist, G-15 and the main estrogenic hormone, 17ß-estradiol were investigated on seizures and brain tissue oxidative damages induced by pentylenetetrazole (PTZ) in rats. METHODS: In this study, 30 adult male Wistar albino rats were used. Due to intraperitoneal (ip) injections of a subconvulsant dose of PTZ (35mg/kg) which was repeated 12 times every 48h, chemical kindling occurred and kindling seizure was recorded for 30min. The rats were injected with 17ß-estradiol (5µg/kg, ip) or G-1 (5µg/kg, ip), G-15 (5µg/kg, ip), Saline, Ethanol and Dimethyl sulfoxide (DMSO) 30min before each dose of PTZ. Observed seizures were classified between the phase 0-5. Thirty minutes later when the last 12. PTZ administration, all rats were sacrificed and the brain cortex, hippocampus sections were removed and the tissue superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) levels on these tissues were studied. RESULTS: GPER1 agonist, G-1 and estrogenic hormone, 17ß-estradiol significantly increased the development of PTZ kindling the seizures. However, GPER1 antagonist, G-15 did not change the development of PTZ kindling the seizures. In the cortex and hippocampus homogenates, the NO levels after G-1 administration had significantly increased (p<0.05) compared to the PTZ groups but SOD activities and MDA levels demonstrated no difference between the groups. CONCLUSIONS: This is the first study that explores that GPER-1 receptors have epileptogenic effect on PTZ-induced kindling rat. GPER1 may mediate the epileptogenic effect of estrogens by changing the oxidative or anti-oxidative parameters in the brain.

Soluble epoxide hydrolase activity regulates inflammatory responses and seizure generation in two mouse models of temporal lobe epilepsy.

Neuroinflammation is known to be involved in epileptogenesis with unclear mechanisms. Inhibition of soluble epoxide hydrolase (sEH) seems to offer anti-inflammatory protection to ischemic brain injury in rodents. Thus, it is hypothesized that sEH inhibition might also affect the neuroinflammatory responses caused by epileptic seizures. In the present study, we investigated the involvement of sEH in neuroinflammation, seizure generation and subsequent epileptogenesis using two mouse models of temporal lobe epilepsy. Experimental epileptic seizures were induced by either pilocarpine or electrical amygdala kindling in both wild-type (WT) C57BL/6 mice and sEH knockout (sEH KO) mice. The sEH expression in the hippocampus was detected by immunohistochemistry and Western blot analysis. The effects of the sEH hydrolase inhibitors, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy) phenyl)-urea (TPPU), and of the genetic deletion of sEH on seizure-induced neuroinflammatory responses and the development of epilepsy were evaluated. In the hippocampus of WT mice, sEH was mainly expressed in astrocytes (GFAP(+)), neurons (NeuN(+)) and scattered microglia (Iba-1(+)) in the regions of CA1, CA3 and dentate gyrus. Expression of sEH was significantly increased on day 7, 14, 21 and 28 after pilocarpine-induced status epilepticus (SE). Administration with sEH inhibitors attenuated the SE-induced up-regulation of interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), the degradation of EETs, as well as IκB phosphorylation. Following treatment with AUDA, the frequency and duration of spontaneous motor seizures in the pilocarpine-SE mice were decreased and the seizure-induction threshold of the fully kindled mice was increased. Up-regulation of hippocampal IL-1ß and IL-6 was found in both WT and sEH KO mice after successful induction of SE. Notably, sEH KO mice were more susceptible to seizures than WT mice. Seizure related neuroinflammation and ictogenesis were attenuated by pharmacological inhibition of sEH enzymatic activity but not by sEH genetic deletion. Therefore, sEH may play an important role in the generation of epilepsy. Furthermore, the effectiveness of AUDA in terms of anti-inflammatory and anti-ictogenesis properties suggests that it may have clinical therapeutic implication for epilepsy in the future, particularly when treating temporal lobe epilepsy.

Effect of edaravone on apoptosis of hippocampus neuron in seizures rats kindled by pentylenetetrazole.

OBJECTIVES: To explore the effect of edaravone (ED) on apoptosis of hippocampus neurons in seizures rats induced by pentylenetetrazole (PTZ). MATERIALS AND METHODS: Forty-eight adult Wistar rats were randomly divided into normal control (NC) group, PTZ group, and ED group. A dose of PTZ [35 mg/(kg·day)] was intraperitoneally (i.p.) injected into the rats of PTZ group and ED group until the kindling criterion was reached. After kindling, the rats of ED group were administered i.p. with ED [0.8 mg/(kg·day)]; the rats of PTZ group were administered i.p. with normal saline. After 30 min, seizures were induced by administering PTZ i.p. The influence of ED on Fas, Caspase-3, and Survivin protein immunoreactivity on hippocampus neurons was studied by immunohistochemistry method. RESULTS: Fas and Caspase-3 positive cells and optical density in hippocampus in PTZ group were more than that of ED group and NC group (all p < 0.01), but Survivin-positive cells and optical density in hippocampus in PTZ group were less than that of ED group (p < 0.01) and were more than that of NC group (p < 0.05); Fas- and Caspase-3-positive cells and optical density in hippocampus in ED group were more than that of NC group (all p < 0.05), but Survivin-positive cells and optical density in hippocampus in ED group were more than that of NC group (p < 0.01). CONCLUSIONS: Seizure can induce apoptosis of hippocampus neurons on seizures rats, but ED can resist the apoptosis of hippocampus neurons by increased expression of Survivin and decreased expression of Fas and Caspase-3.

[Expression of δ subunit of γ-aminobutyric acid A in hippocampus induced by insular-kindled rats].

OBJECTIVE: To explore the expression of δ subunit-containing γ-aminobutyric acid(A) (GABA)(A) receptors in insular electrical kindled rats and analyze the significance of the findings. METHODS: A total of 48 male Sprague-Dawley rats were divided randomly into 3 groups: kindled, sham-operated and control. Kindled group: chronic insular electrical kindled models of rats established by electrical stimulation. Sham-operated group: the same method of the kindled group without electrical stimulation. CONTROL GROUP: without surgery. The number of hippocampal neurons of rat epilepsy, sham-operated and control groups were detected by Nissl staining. The hippocampal expressions of δ subunit of these groups were tested by immunohistochemistry. The mRNA of δ subunit of hippocampus of these groups was detected by quantitative-polymerase chain reaction (q-PCR). RESULTS: The findings of Nissl staining showed that there was no significant difference of the number of hippocampal neurons between epileptic and normal rats (P > 0.05). The findings of q-PCR showed that mRNA of δ subunit of the hippocampus of sham-operated and control groups represented much more than that of kindled group at Day 7 post-kindling (P < 0.01). The findings of immunohistochemistry showed that the hippocampal expression of δ subunit of sham-operated and control groups represented much more than that of kindled group at Day 7 post-kindling (P < 0.01). CONCLUSION: The hippocampal expression of δ subunit-containing GABA(A) receptors decreases in insular epilepsy. It may play an important role in the occurrence and development of insular epilepsy.

The effect of PTZ-induced epileptic seizures on hippocampal expression of PSA-NCAM in offspring born to kindled rats.

BACKGROUND: Maternal epileptic seizures during pregnancy can affect the hippocampal neurons in the offspring. The polysialylated neural cell adhesion molecule (PSA-NCAM), which is expressed in the developing central nervous system, may play important roles in neuronal migration, synaptogenesis, and axonal outgrowth. This study was designed to assess the effects of kindling either with or without maternal seizures on hippocampal PSA-NCAM expression in rat offspring. METHODS: Forty timed-pregnant Wistar rats were divided into four groups: A) Kind+/Seiz+, pregnant kindled (induced two weeks prior to pregnancy) rats that received repeated intraperitoneal (i.p.) pentylenetetrazol, PTZ injections on gestational days (GD) 14-19; B) Kind-/Seiz+, pregnant non-kindled rats that received PTZ injections on GD14-GD19; C) Kind+/Seiz-, pregnant kindled rats that did not receive any PTZ injections; and D) Kind-/Seiz-, the sham controls. Following birth, the pups were sacrificed on PD1 and PD14, and PSA-NCAM expression and localization in neonates' hippocampi were analyzed by Western blots and immunohistochemistry. RESULTS: Our data show a significant down regulation of hippocampal PSA-NCAM expression in the offspring of Kind+/Seiz+ (p = 0.001) and Kind-/Seiz+ (p = 0.001) groups compared to the sham control group. The PSA-NCAM immunoreactivity was markedly decreased in all parts of the hippocampus, especially in the CA3 region, in Kind+/Seiz+ (p = 0.007) and Kind-/Seiz+ (p = 0.007) group's newborns on both PD1 and 14. CONCLUSION: Our findings demonstrate that maternal seizures but not kindling influence the expression of PSA-NCAM in the offspring's hippocampi, which may be considered as a factor for learning/memory and cognitive impairments reported in children born to epileptic mothers.

Combined gene overexpression of neuropeptide Y and its receptor Y5 in the hippocampus suppresses seizures.

We recently demonstrated that recombinant adeno-associated viral vector-induced hippocampal overexpression of neuropeptide Y receptor, Y2, exerts a seizure-suppressant effect in kindling and kainate-induced models of epilepsy in rats. Interestingly, additional overexpression of neuropeptide Y in the hippocampus strengthened the seizure-suppressant effect of transgene Y2 receptors. Here we show for the first time that another neuropeptide Y receptor, Y5, can also be overexpressed in the hippocampus. However, unlike Y2 receptor overexpression, transgene Y5 receptors in the hippocampus had no effect on kainate-induced motor seizures in rats. However, combined overexpression of Y5 receptors and neuropeptide Y exerted prominent suppression of seizures. This seizure-suppressant effect of combination gene therapy with Y5 receptors and neuropeptide Y was significantly stronger as compared to neuropeptide Y overexpression alone. These results suggest that overexpression of Y5 receptors in combination with neuropeptide Y could be an alternative approach for more effective suppression of hippocampal seizures.

[mRNA expression of activity-regulated cytoskeletal-associated protein in hippocampus induced by insular-kindled rats].

OBJECTIVE: To investigate the expression of Arc mRNA in insular electrical kindled and single electrical stimulated rats and its significance. METHODS: Male SD rats were divided randomly into kindled group, single electrical stimulated group, sham-operated group and control group. Each group was divided into 2 sub-groups at different time points. Kindled group: establishing chronic insular electrical kindled model, decapitation to perform RT-PCR of Arc mRNA on hippocampus and to applying in situ hybridization of Arc mRNA on dentate gyrus; Single electrical stimulated group: using the same method as the kindled group with only electrical stimulation twice; Sham-operated group: using the same method to the kindled group without electrical stimulation; CONTROL GROUP: no surgery. RESULTS: Expression of Arc mRNA in the hippocampus of single electrical stimulated, sham-operated and control groups were 0.72 ± 0.14, 0.75 ± 0.16 and 0.71 ± 0.14 respectively. They were significantly less than the kindled group of 1.78 ± 0.43(P < 0.01) at 3 h. The expression of Arc mRNA had no significant difference (P > 0.05) among 4 groups at 6 h; in situ hybridization (cell count) of Arc mRNA showed the expression of Arc mRNA in the kindled group was 112.8 ± 6.0. And it was significantly higher than the other three groups of 46.25 ± 4.35, 45.25 ± 6.23, 44.75 ± 6.49 (P < 0.01) after 3 h, there were no significant difference among 4 groups at 6 h (P > 0.05). CONCLUSION: Insular epilepsy increase the expression of Arc mRNA in hippocampus. Arc may play an important role in the synaptic plasticity of insular epilepsy.

Effect of intravenous immunoglobulin treatment on brain interferon-gamma and interleukin-6 levels in a rat kindling model.

Many studies indicate that intravenous immunoglobulin (IgG) therapy may decrease symptoms of epilepsy. In this study, we assessed the effects of intravenous IgG in an experimental rat kindling model and attempted to elucidate the underlying mechanism of the IgG effect. For induction of kindling, Wistar rats received repeated intraperitoneal injections of picrotoxin. The serum level of neuron-specific enolase (NSE) was measured to determine seizure severity. Interferon (IFN)-gamma and interleukin (IL)-6 levels were measured in rat hippocampus homogenates. The serum NSE level and hippocampal IFN-gamma level were significantly higher in fully kindled, untreated rats compared to unkindled control rats, whereas IL-6 levels were similar in all groups. Intravenous IgG-treated kindled rats showed NSE and IFN-gamma levels similar to those of control rats, along with lower seizure severity and longer seizure latent period than fully kindled, untreated rats. These results indicate that intravenous immunoglobulin exerts a protective effect on the neurons of kindled rats, potentially by downregulating cytokines in the brain. These results shed light on the mechanism by which intravenous immunoglobulin decreases the severity of epileptic seizures.

Benefits and risks of intranigral transplantation of GABA-producing cells subsequent to the establishment of kindling-induced seizures.

Neural transplantation has been investigated experimentally and clinically for the purpose of developing new treatment options for intractable epilepsy. In the present study we assessed the anticonvulsant efficacy and safety of bilateral allotransplantation of genetically engineered striatal GABAergic rat cell lines into the substantia nigra pars reticulata (SNr). Rats with previously-established seizures, induced by amygdala kindling, were used as a model of temporal lobe epilepsy. Three cell lines were transplanted: (1) immortalized GABAergic cells (M213-2O) derived from embryonic rat striatum; (2) M213-2O cells (CL4) transfected with human GAD67 cDNA to obtain higher GABA synthesis than the parent cell line; and (3) control cells (121-1I), also derived from embryonic rat striatum, but which did not show GAD expression. A second control group received injections of medium alone. Transplantation of M213-2O cells into the SNr of kindled rats resulted in significant but transient anticonvulsant effects. Neither control cells nor medium induced anticonvulsant effects. Strong tissue reactions were, however, induced in the host brain of kindled but not of non-kindled rats, and only in animals that received grafts of genetically modified CL4 cells. These tissue reactions included graft rejection, massive infiltration of inflammatory immune cells, and gliosis. The anticonvulsant effect of M213-2O cells emphasizes the feasibility of local manipulations of seizures by intranigral transplantation of GABA-producing cells. On the other hand, the present data suggest that kindling-induced activation of microglia in the SNr can enhance immune reactions to transplanted cells. In this case, under conditions of further immunological stimulation by CL4 cells, transfected with a human cDNA, substantial immune reactions occurred. Thus, it appears that the condition of the host brain and the production of foreign proteins by transplanted cells have to be considered in estimating the risks of rejection of transplants into the brain.

The effect of progesterone on total brain tissue sialic acid levels in experimental epilepsy.

Epilepsy, the most common neurological disorder worldwide, changing cellular interactions and connectivity may have effects on sialic acid levels. A total of 80 mice were separated into 8 groups: the sham, control, pentylentetrazole (PTZ), PTZ plus progesterone, five dose progesterone, single dose progesterone, kindling, and kindling plus progesterone groups. Brains of each mice were extracted and were divided into five parts. The sialic acid levels were significantly different between the groups and also in the subgroups. The results suggested that progesterone may have an anti-seizure effect by decreasing sialic acid levels in mice. Further studies are needed to evaluate the role of progesterone on sialic acid levels and its role in the epilepsy pathogenesis.

[Effect of carbenoxolone on expression of Fos, NMDAR2 and GFAP in the hippocampus of pentylenetetrazo-kindled epileptic rats].

OBJECTIVE: Gap junctions, the clusters of intercellular channels, play an important role in synchronizing electrical activity. This study investigated the effect of gap junction blocker carbenoxolone (CBX) on epileptic activity in pentylenetetrazo (PTZ)-kindled rats. METHODS: Thirty adult male SD rats were randomly divided into three groups: control, PTZ-kindled and CBX-treated groups (n=10 each). The rats from the PTZ-kindled and the CBX-treated groups were intraperitoneally injected with PTZ (35 mg/kg x d) to induce epilepsy. After epilepsy kindling, they were intraperitoneally injected for 3 days with CBX (10 mg/kg) (CBX-treated group) or with normal saline (PTZ-kindled group). The control group received intraperitoneal injections of normal saline. Anti-GFAP, anti-Fos, and anti-NMDARZ immunohistochemical ABC methods were used to detect the expression of GFAP-Li, Fos-Li and NMDAR2-Li in the hippocampus respectively. RESULTS: Spontaneous seizures occurred in PTZ-kindled epileptic rats. CBX administration reduced spontaneous seizures. The NMDAR2-Li and Fos-Li neurons as well as GFAP-Li astrocytes in hippocampi increased in PTZ-kindled epileptic rats compared with controls. The numbers of Fos-Li (93.75 +/-7.94 vs 165.25 +/-15.87, P < 0.05) and NMDAR2-Li neurons (61.47 +/-3.62 vs 148.72 +/-14.53, P < 0.01) in the CBX-treated group were significantly less than in the PTZ-kindled group. There were no significant differences in the GFAP-Li expression between the CBX-treated and the PTZ-kindled groups. CONCLUSIONS: CBX may inhibit spontaneous seizures and decrease the numbers of Fos-Li and NMDARZ-Li neurons, thus providing anti-epileptic effects.

Modified variant of the Rieske iron-sulfur protein in the hippocampus of kindled rats and human epileptic patients.

Kindled seizures are widely used to model epileptogenesis, but the molecular mechanisms underlying the attainment of kindling status are largely unknown. Recently we showed that achievement of kindling status in the Sprague-Dawley rat is associated with a critical developmental interval of 25 +/- 1 days; the identification of this long, well-defined developmental interval for inducing kindling status makes possible a dissection of the cellular and genetic events underlying this phenomenon and its relationship to normal and pathological brain function. Now we report the identification, by proteomics, of a modified variant of the Rieske iron-sulfur protein, a component of the mitochondrial cytochrome bc1 complex, whose isoelectric point is shifted toward more alkaline values in the hippocampus of kindled rats. By immunohistochemistry the Rieske protein is well-expressed in the hippocampus except in the CA1 subfield, a region of selective vulnerability to seizures in humans and animal models. We also noted an asymmetric, selective expression of the Rieske protein in the subgranular neurons of the dorsal dentate gyrus, a region implicated in neurogenesis. Abnormal changes in Rieske protein immunoreactivity also were found in sections obtained from human epileptic patients. These results suggest that the Rieske protein may play a role in the response of neurons to seizure activity and could give important new insights into the molecular pathogenesis of epilepsy.

Suppression of kindling epileptogenesis by adenosine releasing stem cell-derived brain implants.

Epilepsy therapy is largely symptomatic and no effective therapy is available to prevent epileptogenesis. We therefore analysed the potential of stem cell-derived brain implants and of paracrine adenosine release to suppress the progressive development of seizures in the rat kindling-model. Embryonic stem (ES) cells, engineered to release the inhibitory neuromodulator adenosine by biallelic genetic disruption of the adenosine kinase gene (Adk-/-), and respective wild-type (wt) cells, were differentiated into neural precursor cells (NPs) and injected into the hippocampus of rats prior to kindling. Therapeutic effects of NP-derived brain implants were compared with those of wt baby hamster kidney cells (BHK) and adenosine releasing BHK cell implants (BHK-AK2), which were previously shown to suppress seizures by paracrine adenosine release. Wild-type NP-graft recipients were characterized by an initial delay of seizure development, while recipients of adenosine releasing NPs displayed sustained protection from developing generalized seizures. In contrast, recipients of wt BHK cells failed to display any effects on kindling development, while recipients of BHK-AK2 cells were only moderately protected from seizure development. The therapeutic effect of Adk(-/-)-NPs was due to graft-mediated adenosine release, since seizures could transiently be provoked after blocking adenosine A1 receptors. Histological analysis of NP-implants at day 26 revealed cell clusters within the infrahippocampal cleft as well as intrahippocampal location of graft-derived cells expressing mature neuronal markers. In contrast, BHK and BHK-AK2 cell implants only formed cell clusters within the infrahippocampal cleft. We conclude that ES cell-derived adenosine releasing brain implants are superior to paracrine adenosine release from BHK-AK2 cell implants in suppressing seizure progression in the rat kindling-model. These findings may indicate a potential antiepileptogenic function of stem cell-mediated adenosine delivery.

The anticonvulsant effects of progesterone and its metabolites on amygdala-kindled seizures in male rats.

Progesterone is a neurosteroid that modulates neuronal excitability. The anticonvulsant effects of progesterone are largely mediated by the actions of its metabolites. The purpose of this study was to measure the anticonvulsant effects of progesterone, 5alpha-dihydroprogesterone, and allopregnanolone against amygdala-kindled seizures in male rats. The amygdala kindling model is a model of human complex partial seizures with secondary generalization. A bipolar electrode was chronically implanted in the right amygdala of male Wistar rats. All subjects were kindled to 30 stage 5 seizures and stability tested. Multiple doses of progesterone, 5alpha-dihydroprogesterone, or allopregnanolone were administered in separate dose-response studies. The antiseizure effects of each compound were determined. A progesterone time-response study was also conducted. At 30 min after injection, progesterone had an ED50 of 65.3 mg/kg against the secondarily generalized seizure and an ED50 of 114 mg/kg against the focal seizure. 5alpha-dihydroprogesterone had a low ED50 of 6.2 mg/kg against both the generalized component of the amygdala-kindled seizure and the focal seizure. Allopregnanolone had an ED50 of 15.2 mg/kg against the secondarily generalized seizure and was not effective against the focal seizure. Progesterone is an effective anticonvulsant against the secondarily generalized component of amygdala-kindled seizures in male rats. Progesterone is only effective against the focal seizure at high ataxic doses. 5alpha-dihydroprogesterone is a potent anticonvulsant against both the kindled amygdala focal discharge and the secondarily generalized seizure. Allopregnanolone is an effective anticonvulsant against the secondarily generalized component of the seizure, but not against the amygdala focal discharge.