Pilocarpine Model Of Status Epilepticus Research Articles

Increased Susceptibility to Pilocarpine-Induced Status Epilepticus and Reduced Latency in TRPC1/4 Double Knockout Mice.

Canonical transient receptor potential channels (TRPCs) are a family of calcium-permeable cation channels. Previous studies have shown that heteromeric channels comprising TRPC1 and TRPC4 mediate epileptiform bursting in lateral septal neurons and hippocampal CA1 pyramidal neurons, suggesting that TRPC1/4 channels play a pro-seizure role. In this study, we utilized electroencephalography (EEG) recording and spectral analysis to assess the role of TRPC1/4 channels in the pilocarpine model of status epilepticus (SE). We found that, surprisingly, TRPC1/4 double knockout (DKO) mice exhibited an increased susceptibility to pilocarpine-induced SE. Furthermore, SE latency was also significantly reduced in TRPC1/4 DKO mice. Further studies are needed to reveal the underlying mechanisms of our unexpected results.

Modulation of neuropathology and cognitive deficits by lipopolysaccharide preconditioning in a mouse pilocarpine model of status epilepticus

Recent studies indicate that microglia may play a critical role in epileptogenesis during the early post-status epilepticus (SE) period. In this study, we aimed to elucidate the effects of preconditioning of microglia with lipopolysaccharide (LPS) on neuropathology and cognitive deficits in a mouse pilocarpine model of SE. Mice were treated with an intraperitoneal injection of LPS 24 h before SE induction. The open field test at 13 days after SE showed that LPS preconditioning suppressed SE-induced hyperactivity. The Y-maze test at 14 days after SE showed that LPS preconditioning ameliorated SE-induced working memory impairment. The extent of neuronal damage was decreased by LPS preconditioning in the hippocampus of mice euthanized at 15 days after SE. Gene profile analysis of hippocampal microglia at 15 days after SE showed that the expression level of interleukin-1β was increased by SE induction but decreased by LPS preconditioning. By contrast, SE induction increased the expression levels of phagocytosis-related genes, and LPS preconditioning further enhanced their expression. Interestingly, LPS preconditioning increased the numerical density of hippocampal microglia expressing the 5D4 keratan sulfate epitope, a population of cells known to be involved in phagocytosis. The voxel density of glutamatergic synapses was increased by SE induction but decreased by LPS preconditioning, while GABAergic synapses were not affected by these procedures. Our findings indicate that LPS preconditioning may in part alleviate SE-related abnormal synaptogenesis and cognitive deficits, and also suggest that modulation of microglial activation during the early post-SE period may be a novel strategy for epilepsy treatment.

Systemic thrombin inhibition ameliorates seizures in a mouse model of pilocarpine-induced status epilepticus.

Status epilepticus (SE) is a life-threatening condition characterized by ongoing seizure activity which can lead to severe brain damage and death if not treated properly. Recent work suggests that alterations in blood-brain barrier (BBB) function and subsequent cortical exposure to coagulation factors may initiate, promote, and/or sustain SE. This suggestion is based on the observation that the serine protease thrombin, which plays a fundamental role in the blood coagulation cascade, increases neural excitability through the activation of protease-activated receptor 1 (PAR1). However, it remains unclear whether systemic inhibition of thrombin asserts "anti-epileptic" effects in vivo. We here used the pilocarpine model of SE in adult 3-month-old male mice to address the question whether intraperitoneal injection of the thrombin inhibitor α-NAPAP (0.75 mg/kg) counters SE. Indeed, pharmacological inhibition of thrombin ameliorates the behavioral outcome of pilocarpine-induced SE. Similar results are obtained when the thrombin receptor PAR1 is pharmacologically blocked using intraperitoneal injection of SCH79797 (25 μg/kg) prior to SE induction. Consistent with these results, an increase in thrombin immunofluorescence is detected in the hippocampus of pilocarpine-treated animals. Moreover, increased hippocampal serine protease activity is detected 90 min after SE induction, which is not observed in animals treated with α-NAPAP prior to SE induction. Together, these results corroborate and extend recent studies suggesting that novel oral anticoagulants which target thrombin (and PAR1) may assert anti-epileptic effects in vivo. KEY MESSAGES: Systemic thrombin/PAR1-inhibition ameliorates anticoagulants behavioral seizures. Status epilepticus increases thrombin levels in the hippocampus. Increased serine protease activity in the hippocampus after status epileptic. Anti-epileptic potential of clinically used anticoagulants must be evaluated.

Extrasynaptic γ-aminobutyric acid type A receptor-mediated sex differences in the antiseizure activity of neurosteroids in status epilepticus and complex partial seizures.

Sex differences are evident in the antiseizure activity of neurosteroids; however, the potential mechanisms remain unclear. In this study, we sought to determine whether differences in target extrasynaptic δ-subunit γ-aminobutyric acid type A (GABA-A) receptor expression and function underlie the sex differences in seizure susceptibility and the antiseizure activity of neurosteroids. Sex differences in seizure susceptibility and protective activity of three distinct neurosteroids-allopregnanolone (AP), androstanediol (AD), and ganaxolone-were evaluated in the pilocarpine model of status epilepticus (SE) and kindling seizure test in mice. Immunocytochemistry was used for δGABA-A receptor expression analysis, and patch-clamp recordings in brain slices evaluated its functional currents. Sex differences were apparent in kindling epileptogenic seizures, with males exhibiting a faster progression to a fully kindled state. Neurosteroids AP, AD, or ganaxolone produced dose-dependent protection against SE and acute partial seizures. However, female mice exhibited strikingly enhanced sensitivity to the antiseizure activity of neurosteroids compared to males. Sex differences in neurosteroid protection were unrelated to pharmacokinetic factors, as plasma levels of neurosteroids associated with seizure protection were similar between sexes. Mice lacking extrasynaptic δGABA-A receptors did not exhibit sex differences in neurosteroid protection. Consistent with a greater abundance of extrasynaptic δGABA-A receptors, AP produced a significantly greater potentiation of tonic currents in dentate gyrus granule cells in females than males; however, such enhanced AP sensitivity was diminished in δGABA-A receptor knockout female mice. Neurosteroids exhibit greater antiseizure potency in females than males, likely due to a greater abundance of extrasynaptic δGABA-A receptors that mediate neurosteroid-sensitive tonic currents and seizure protection. These findings indicate the potential to develop personalized gender-specific neurosteroid treatments for SE and epilepsy in men and women, including catamenial epilepsy.

The effects of calcineurin inhibitor FK506 on actin cytoskeleton, neuronal survival and glial reactions after pilocarpine-induced status epilepticus in mice

After status epilepticus (SE), actin cytoskeleton (F-actin) becomes progressively deconstructed in the hippocampus, which is consistent with the delayed pyramidal cell death in both time course and spatial distribution. A variety of experiments show that calcineurin inhibitors such as FK506 are able to inhibit the SE-induced actin depolymerization. However, it is still unclear what changes happen to the F-actin in the epileptic brain after FK506 treatment. A pilocarpine model of SE in mice was used to examine the effects of FK506 on the F-actin in the hippocampal neurons. The post SE (PSE) mice with or without FK506 treatment were monitored consecutively for 14 days to examine the frequency and duration of spontaneous seizures. The effects of FK506 on the activity of cofilin and actin dynamics were assessed at 7 and 14 d PSE by western blots. The organization of F-actin, neuronal cell death, and glial reactions were investigated by phalloidin staining, histological and immunocytochemical staining, respectively. As compared to the PSE + vehicle mice, FK506 treatment significantly decreased the frequency and duration of spontaneous seizures. Relative to the PSE + vehicle mice, western blots detected a partial restoration of phosphorylated cofilin and a significant increase of F/G ratio in the hippocampus after FK506 treatment. In the PSE + vehicle mice, almost no F-actin puncta were left in the CA1 and CA3 subfields at 7 and 14 d PSE. FK506-treated PSE mice showed a similar decrease of F-actin, but the extent of damage was significantly ameliorated. Consistently, the surviving neurons became significantly increased in number after FK506 treatment, relative to the PSE + vehicle groups. After FK506 treatment, microglial reaction was partially inhibited, but the expression of GFAP was not significantly changed, compared to the PSE + vehicle mice. The results suggest that post-epileptic treatment with FK506 ameliorated, but could not stop the deconstruction of F-actin or the delayed neuronal loss in the PSE mice.

Molecular imaging reveals epileptogenic Ca2+-channel promoter activation in hippocampi of living mice.

Focal epilepsies often originate in the hippocampal formation of the temporal lobe (temporal lobe epilepsy) and are generally acquired after transient brain insults. Such insults induce cellular and structural reorganization processes of the hippocampus, referred to as epileptogenesis that finally convert the brain spontaneous epileptic. Here, we developed a new molecular imaging strategy in a state-of-the-art animal model to provide insights into key epileptogenic mechanisms. Our new approach combines recombinant adeno-associated virus (rAAV) gene delivery with in vivo bioluminescence imaging. rAAV particles harboring the luciferase reporter gene under control of the minimal T type Ca(2+)-channel subunit Ca V 3.2-promoter were generated and injected stereotaxically in the hippocampal region of mice. Bioluminescent signals, corresponding to Ca V 3.2 promoter activation, were imaged in vivo in the pilocarpine model of status epilepticus (SE). We detected activation of key Ca V 3.2 promoter motifs at 3 and 10 days after SE but not after the onset of chronic seizures. These data suggest Ca V 3.2 promoter activation as novel anti-epileptogenic target. In more general terms, we have established an experimental approach that allows to follow cerebral gene promoter dynamics longitudinally and to correlate this activity to behavioral parameters in the same mice.

Protective but Not Anticonvulsant Effects of Ghrelin and JMV-1843 in the Pilocarpine Model of Status epilepticus

In models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagogue-receptor 1a (GHS-R1a). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45±0.07 mm2 in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P<0.05) and JMV-1843 (P<0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P<0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.

Inhibition of the prostaglandin receptor EP2 following status epilepticus reduces delayed mortality and brain inflammation

Prostaglandin E2 is now widely recognized to play critical roles in brain inflammation and injury, although the responsible prostaglandin receptors have not been fully identified. We developed a potent and selective antagonist for the prostaglandin E2 receptor subtype EP2, TG6-10-1, with a sufficient pharmacokinetic profile to be used in vivo. We found that in the mouse pilocarpine model of status epilepticus (SE), systemic administration of TG6-10-1 completely recapitulates the effects of conditional ablation of cyclooxygenase-2 from principal forebrain neurons, namely reduced delayed mortality, accelerated recovery from weight loss, reduced brain inflammation, prevention of blood-brain barrier opening, and neuroprotection in the hippocampus, without modifying seizures acutely. Prolonged SE in humans causes high mortality and morbidity that are associated with brain inflammation and injury, but currently the only effective treatment is to stop the seizures quickly enough with anticonvulsants to prevent brain damage. Our results suggest that the prostaglandin receptor EP2 is critically involved in neuroinflammation and neurodegeneration, and point to EP2 receptor antagonism as an adjunctive therapeutic strategy to treat SE.

Corticotropin releasing factor (CRF) in the hippocampus of the mouse pilocarpine model of status epilepticus

We investigated the cellular localization and progressive changes of corticotropin releasing factor (CRF) in the mouse hippocampus, during and after pilocarpine induced status epilepticus (PISE) and subsequent epileptogenesis. We found that CRF gene expression was up-regulated significantly at 2h during and 1d after PISE in comparison to control mice. Immunohistochemical analysis showed that the number of CRF and Fos immunoreactive cells was increased significantly in the strata oriens and pyramidale of CA1 area and in the stratum pyramidale of CA3 area at 2h during and 1d after PISE. CRF was induced in calbindin (CB) or calretinin (CR) immunoreactive interneurons in stratum oriens at 2h during PISE. It suggests that induced CRF may be related to the over excitation of hippocampal neurons and occurrence of status epilepticus. It may also cause excitoneurotoxicity and delayed loss of CA3 and CA1 pyramidal neurons, leading to the onset of epilepsy.

Excitatory Input Onto Hilar Somatostatin Interneurons Is Increased in a Chronic Model of Epilepsy

The density of somatostatin (SOM)-containing GABAergic interneurons in the hilus of the dentate gyrus is significantly decreased in both human and experimental temporal lobe epilepsy. We used the pilocarpine model of status epilepticus and temporal lobe epilepsy in mice to study anatomical and electrophysiological properties of surviving somatostatin interneurons and determine whether compensatory functional changes occur that might offset loss of other inhibitory neurons. Using standard patch-clamp techniques and pipettes containing biocytin, whole cell recordings were obtained in hippocampal slices maintained in vitro. Hilar SOM cells containing enhanced green fluorescent protein (EGFP) were identified with fluorescent and infrared differential interference contrast video microscopy in epileptic and control GIN (EGFP-expressing Inhibitory Neurons) mice. Results showed that SOM cells from epileptic mice had 1) significant increases in somatic area and dendritic length; 2) changes in membrane properties, including a small but significant decrease in resting membrane potential, and increases in time constant and whole cell capacitance; 3) increased frequency of slowly rising spontaneous excitatory postsynaptic currents (sEPSCs) due primarily to increased mEPSC frequency, without changes in the probability of release; 4) increased evoked EPSC amplitude; and 5) increased spontaneous action potential generation in cell-attached recordings. Results suggest an increase in excitatory innervation, perhaps on distal dendrites, considering the slower rising EPSCs and increased output of hilar SOM cells in this model of epilepsy. In sum, these changes would be expected to increase the inhibitory output of surviving SOM interneurons and in part compensate for interneuronal loss in the epileptogenic hippocampus.

Homocysteine Potentiates Seizures and Cell Loss Induced by Pilocarpine Treatment

Patients affected by recurrent seizures frequently present increased homocysteine plasma levels in consequence of treatment with antiepileptic drugs. Homocysteine is proconvulsant and can affect the response to antiepileptic drugs. In addition, high homocysteine plasma levels represent a risk factor for cardiovascular and neurodegenerative diseases. To better define the role of increased homocysteine in epilepsy, we analyzed the effects of homocysteine pretreatment in the pilocarpine model of status epilepticus (SE), which is used to mimic temporal lobe epilepsy (TLE) in rodents. Precisely, we investigated whether a moderate hyperhomocysteinemia, unable to cause seizures, could sensitize rats to pilocarpine and cooperate in inducing brain lesions. We found that a subthreshold dose of pilocarpine (200 mg/kg) is sufficient to induce SE in the majority (approximately 90%) of rats pretreated with homocysteine for 2 weeks, whereas only 40% of saline-treated controls developed SE following the same pilocarpine dose. Furthermore, homocysteine pretreatment led to a significant increase in neuronal cell loss evaluated by counting toluidine blue-stained or Fluoro-Jade-positive cells in hippocampal and parahippocampal regions. Pilocarpine augmented amyloid beta expression in both animal groups. However, pretreatment with homocysteine favored the intraneuronal fibrillar conformation of amyloid beta, thus promoting neurodegeneration. These findings indicate that increased homocysteine levels enhance seizure activity and neurodegeneration in pilocarpine-treated rats and suggest that similar detrimental effects may occur in patients affected by TLE.

Prolonged seizure activity leads to increased Protein Kinase A activation in the rat pilocarpine model of status epilepticus

Status epilepticus is a life-threatening form of seizure activity that represents a major medical emergency associated with significant morbidity and mortality. Protein Kinase A is an important regulator of synaptic strength that may play an important role in the development of status epilepticus-induced neuronal pathology. This study demonstrated an increase in PKA activity against exogenous and endogenous substrates during later stages of SE. As SE progressed, a significant increase in PKA-mediated phosphorylation of an exogenous peptide substrate was demonstrated in cortical structures. The increased activity was not due to altered expression of either regulatory or catalytic subunits of the enzyme. Through the use of phospho-specific antibodies, this study also investigated the effects of SE on the phosphorylation of the GluR1 subunit of the AMPA subtype of glutamate receptor. After the onset of continuous seizure activity, an increase in phosphorylation of the PKA site on the GluR1 subunit of the AMPA receptor was observed. These data suggest a potential mechanism by which SE may increase neuronal excitability in the cortex, potentially leading to maintenance of seizure activity or long-term neuronal pathology.

The Spine Loss Paradox: Clues to Mechanisms and Meaning

A Cellular Mechanism for Dendritic Spine Loss in the Pilocarpine Model of Status Epilepticus. Kurz JE, Moore BJ, Henderson SC, Campbell JN, Churn SB. Epilepsia 2008 May 8. [Epub ahead of print] PURPOSE: Previous studies have documented a synaptic translocation of calcineurin (CaN) and increased CaN activity following status epilepticus (SE); however, the cellular effect of these changes in CaN in the pathology of SE remains to be elucidated. This study examined a CaN-dependent modification of the dendritic cytoskeleton. CaN has been shown to induce dephosphorylation of cofilin, an actin depolymerization factor. The ensuing actin depolymerization can lead to a number of physiological changes that are of interest in SE. METHODS: SE was induced by pilocarpine injection, and seizure activity was monitored by video-EEG. Subcellular fractions were isolated by differential centrifugation. CaN activity was assayed using a paranitrophenol phosphate (pNPP) assay protocol. Cofilin phosphorylation was assessed using phosphocofilin-specific antibodies. Cofilin–actin binding was determined by coimmunoprecipitation, and actin polymerization was measured using a triton-solubilization protocol. Spines were visualized using a single-section rapid Golgi impregnation procedure. RESULTS: The immunoreactivity of phosphocofilin decreased significantly in hippocampal and cortical synaptosomal samples after SE. SE-induced cofilin dephosphorylation could be partially blocked by the preinjection of CaN inhibitors. Cofilin activation could be further demonstrated by increased actin–cofilin binding and a significant depolymerization of neuronal actin, both of which were also blocked by CaN inhibitors. Finally, we demonstrated a CaN-dependent loss of dendritic spines histologically. DISCUSSION: The data demonstrate a CaN-dependent, cellular mechanism through which prolonged seizure activity results in loss of dendritic spines via cofilin activation. Further research into this area may provide useful insights into the pathology of SE and epileptogenic mechanisms.

A cellular mechanism for dendritic spine loss in the pilocarpine model of status epilepticus

Previous studies have documented a synaptic translocation of calcineurin (CaN) and increased CaN activity following status epilepticus (SE); however, the cellular effect of these changes in CaN in the pathology of SE remains to be elucidated. This study examined a CaN-dependent modification of the dendritic cytoskeleton. CaN has been shown to induce dephosphorylation of cofilin, an actin depolymerization factor. The ensuing actin depolymerization can lead to a number of physiological changes that are of interest in SE. SE was induced by pilocarpine injection, and seizure activity was monitored by video-EEG. Subcellular fractions were isolated by differential centrifugation. CaN activity was assayed using a paranitrophenol phosphate (pNPP) assay protocol. Cofilin phosphorylation was assessed using phosphocofilin-specific antibodies. Cofilin-actin binding was determined by coimmunoprecipitation, and actin polymerization was measured using a triton-solubilization protocol. Spines were visualized using a single-section rapid Golgi impregnation procedure. The immunoreactivity of phosphocofilin decreased significantly in hippocampal and cortical synaptosomal samples after SE. SE-induced cofilin dephosphorylation could be partially blocked by the preinjection of CaN inhibitors. Cofilin activation could be further demonstrated by increased actin-cofilin binding and a significant depolymerization of neuronal actin, both of which were also blocked by CaN inhibitors. Finally, we demonstrated a CaN-dependent loss of dendritic spines histologically. The data demonstrate a CaN-dependent, cellular mechanism through which prolonged seizure activity results in loss of dendritic spines via cofilin activation. Further research into this area may provide useful insights into the pathology of SE and epileptogenic mechanisms.

Proteome changes associated with hippocampal MRI abnormalities in the lithium pilocarpine‐induced model of convulsive status epilepticus

Convulsive status epilepticus is associated with subsequent hippocampal damage and development of mesial temporal sclerosis in a subset of individuals. The lithium pilocarpine model of status epilepticus (SE) in the rat provides a model in which to investigate the molecular and pathogenic process leading to hippocampal damage. In this study, a 2-DE-based approach was used to detect proteome changes in the hippocampus, at an early stage (2 days) after SE, when increased T2 values were detectable by magnetic resonance imaging. Gel image analysis was followed by LC-MS/MS identification of protein species that differed in abundance between pilocarpine-treated and control rats. The most significantly up-regulated species in the experimental animals was identified as heat shock 27-kDa protein, in line with findings in humans and in other experimental models of epilepsy. Additional up-regulated species included dihydropyrimidinase-related protein-2, cytoskeletal proteins (alpha-tubulin and ezrin) and dihydropteridine reductase. In summary, the hippocampus of rats subject to pilocarpine-induced SE exhibits specific changes in protein abundance, which likely relate to pathogenic, neuroprotective and neurogenic responses.

Anticonvulsive Effect of a Selective mGluR8 Agonist (S)‐3,4‐Dicarboxyphenylglycine (S‐3,4‐DCPG) in the Mouse Pilocarpine Model of Status Epilepticus

We sought to investigate the anticonvulsive and neuroprotective effect of a selective metabotropic glutamate receptor 8 (mGluR8) agonist (S)-3,4-dicarboxyphenylglycines (S-3,4-DCPG) on pilocarpine-induced status epilepticus (PISE) and subsequent loss of hilar neurons in the dentate gyrus after systemic (intravenous) or local (intracerebroventricular) administration. We compared the difference in granular cell responses after paired-pulse stimulation of the perforant pathway and the sensitivity to local injection of S-3,4-DCPG into the stratum granulosum in the control and mice at 2 months after PISE. We used intravenous, intracerebroventricular, or intrahippocampal administration of S-3,4-DCPG to mice with status epilepticus or temporal lobe epilepsy and neurophysiologic recording of somatic field excitatory postsynaptic potential (sfEPSP) and population spike (PS) of granular cells in response to perforant-pathway stimulation or S-3,4-DCPG treatment. Intracerebroventricular (1.91 micromol) but not systemic administration of S-3,4-DCPG (at doses of 12.5, 50, 100, 200, 400, 800, and 1,200 mg/kg) could control PISE with no neuroprotective effect. In epileptic mice, mGluR8-mediated inhibition of fEPSPs was reduced significantly in granular cell bodies. At doses ranging from 12.5 to 1,200 mg/kg, intravenous administration of S-3,4-DCPG may not be effective in controlling status epilepticus. Down-regulation of mGluR8 may be related to reduced S-3,4-DCPG-mediated inhibition and the subsequent occurrence of spontaneously recurrent seizures.

CRE-mediated transcription and COX-2 expression in the pilocarpine model of status epilepticus

Status epilepticus (SE) triggers neuronal death, reactive gliosis and remodeling of synaptic circuitry, thus leading to profound pathological alterations in CNS physiology. These processes are, in part, regulated by the rapid upregulation of both cytotoxic and cytoprotective genes. One pathway that may couple SE to transcriptionally dependent alterations in CNS physiology is the CREB (cAMP response element-binding protein)/CRE (cAMP response element) cascade. Here, we utilized the pilocarpine model of SE on a mouse strain transgenic for a CRE-reporter construct (beta-galactosidase) to begin to characterize how seizure activity regulates the activation state of the CREB/CRE pathway in both glia and neurons of the hippocampus. SE triggered a rapid (4-8 h post-SE) but transient increase in CRE-mediated gene expression in the neuronal sublayers. In contrast to neurons, SE induced a lasting increase (up to 20 days) in CRE-mediated transcription in both reactive astrocytes and microglia. CRE-mediated gene expression correlated with expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2). To examine the role of CREB in SE-induced COX-2 expression, we generated a transgenic mouse strain that expresses A-CREB, a potent repressor of CREB-dependent transcription. In these animals, the capacity of SE to stimulate COX-2 expression was markedly attenuated, indicating that CREB is a key intermediate in SE-induced COX-2 expression. Collectively these data show that SE triggers two waves of CREB-mediated gene expression, a transient wave in neurons and a long-lasting wave in reactive glial cells, and that CREB couples SE to COX-2 expression.

Spastin in the human and mouse central nervous system with special reference to its expression in the hippocampus of mouse pilocarpine model of status epilepticus and temporal lobe epilepsy

In the present in situ hybridization and immunocytochemical studies in the mouse central nervous system (CNS), a strong expression of spastin mRNA and protein was found in Purkinje cells and dentate nucleus in the cerebellum, in hippocampal principal cells and hilar neurons, in amygdala, substantia nigra, striatum, in the motor nuclei of the cranial nerves and in different layers of the cerebral cortex except piriform and entorhinal cortices where only neurons in layer II were strongly stained. Spastin protein and mRNA were weakly expressed in most of the thalamic nuclei. In selected human brain regions such as the cerebral cortex, cerebellum, hippocampus, amygdala, substania nigra and striatum, similar results were obtained. Electron microscopy showed spastin immunopositive staining in the cytoplasma, dendrites, axon terminals and nucleus. In the mouse pilocarpine model of status epilepticus and subsequent temporal lobe epilepsy, spastin expression disappeared in hilar neurons as early as at 2 h during pilocarpine induced status epilepticus, and never recovered. At 7 days and 2 months after pilocarpine induced status epilepticus, spastin expression was down-regulated in granule cells in the dentate gyrus, but induced expression was found in reactive astrocytes. The demonstration of widespread distribution of spastin in functionally different brain regions in the present study may provide neuroanatomical basis to explain why different neurological, psychological disorders and cognitive impairment occur in patients with spastin mutation. Down-regulation or loss of spastin expression in hilar neurons may be related to their degeneration and may therefore initiate epileptogenetic events, leading to temporal lobe epilepsy.

Modulation of CaM Kinase II Activity Is Coincident with Induction of Status Epilepticus in the Rat Pilocarpine Model

This study was conducted to characterize the early cellular changes in CaM kinase II activity that occur during the induction of status epilepticus (SE). The pilocarpine model of SE was characterized both behaviorally and electrographically. At specific time points after the first discrete seizure, specific brain regions were isolated for biochemical study. Phosphate incorporation into a CaM kinase II-specific substrate, autocamtide III, was used to determine kinase activity. After the development of SE, the data show an immediate inhibition of both cortical and hippocampal CaM kinase II activity in homogenate, but a delayed inhibition in synaptic kinase activity. The maintenance of synaptic kinase activity was due to a translocation of CaM kinase II protein to the synapse. However, despite the translocation of functional kinase, CaM kinase II activity was not maintained, membrane potential was not restored, and the newly translocated CaM kinase II did not terminate the SE event. Unlike the homogenate samples, in the crude synaptoplasmic membrane (SPM) subcellular fractions, a positive correlation is found between the duration of SE and the inhibition of CaM kinase II activity in both the cortex and hippocampus. The data support the hypothesis that alterations of CaM kinase II activity are involved in the early events of SE pathology.

Age dependence of pilocarpine-induced status epilepticus and inhibition of CaM kinase II activity in the rat

This study was conducted to characterize the post-pubertal developmental aspects on seizure susceptibility and severity as well as calcium/calmodulin protein kinase type II (CaM kinase II) activity in status epilepticus (SE). Thirty- to ninety-day-old rats, in 10-day increments, were studied. This corresponds to a developmental age group that has not received thorough attention. The pilocarpine model of SE was characterized both behaviorally and electrographically. Seven criteria were analyzed for electrographical characterization: seizure severity, SE susceptibility, the average number of discrete seizures, average time until first seizure, average time to SE, average time from first discrete seizure to SE, and death. After 1 h of SE, specific brain regions were isolated for biochemical study. Phosphate incorporation into a CaM kinase II-specific substrate, autocamtide III, was used to determine kinase activity. There was no developmental effect on the average number of discrete seizures, average time until first seizure, average time to SE, average time from first discrete seizure to SE, and death; however, there was a significant effect on SE probability and seizure severity. Once SE was expressed, all animals showed a decrease in both cortical and hippocampal CaM kinase II activities. Conversely, seizure activity in the absence of SE did not result in a decrease in CaM kinase II activity. The data suggest that there is a gradual age-dependent modulation of SE susceptibility and seizure severity within the developmental stages studied. Additionally, once status epilepticus is observed at any age, there is a corresponding SE-induced inhibition of CaM kinase II.