Accumulating data have revealed that abnormal activity of the mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by various factors. We previously reported that pretreatment with perifosine, an inhibitor of Akt (also called protein kinase B), abolishes the rapamycin-induced paradoxical increase of S6 phosphorylation in a rat model induced by kainic acid (KA). Since Akt is an upstream target in the mTOR signaling pathway, we set out to determine whether perifosine has a preventive effect on epileptogenesis. Here, we explored the effect of perifosine on the model of temporal epilepsy induced by KA in rats and found that pretreatment with perifosine had no effect on the severity or duration of the KA-induced status epilepticus. However, perifosine almost completely inhibited the activation of p-Akt and p-S6 both acutely and chronically following the KA-induced status epilepticus. Perifosine pretreatment suppressed the KA-induced neuronal death and mossy fiber sprouting. The frequency of spontaneous seizures was markedly decreased in rats pretreated with perifosine. Accordingly, rats pretreated with perifosine showed mild impairment in cognitive functions. Collectively, this study provides novel evidence in a KA seizure model that perifosine may be a potential drug for use in anti-epileptogenic therapy.
Animals ; Anticonvulsants ; pharmacology ; Brain ; drug effects ; pathology ; Convulsants ; toxicity ; Disease Models, Animal ; Epilepsy, Temporal Lobe ; chemically induced ; pathology ; Kainic Acid ; toxicity ; Male ; Neurons ; drug effects ; pathology ; Phosphorylcholine ; analogs & derivatives ; pharmacology ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Status Epilepticus ; chemically induced ; pathology

OBJECTIVETo study the effect of a microRNA-132 antagonist on lithium-pilocarpine-induced status epilepticus (SE) in young Sprague-Dawley (SD) rats.

METHODSForty-five 3-week-old SD rats were randomly and equally divided into epilepticus model group, microRNA-132 antagonist group, and microRNA-132 antagonist negative control group. The young SD rat model of SE was established using lithium-pilocarpine. For the microRNA-132 antagonist group and the negative control group, pretreatment was performed 24 hours before the model establishment. Behavioral observation was performed to assess the latency of SE and success rate of induction of SE. The scale of Lado was used to evaluate the seizure severity. Electroencephalography (EEG) was used to assess the frequency and amplitude of epileptiform discharges. The mortality rate was calculated in each group.

RESULTSThere was no significant difference in the success rate of induction of SE between the three groups (P>0.05). Compared with the microRNA-132 negative control group and the epilepticus model group, the microRNA-132 antagonist group had significantly prolonged SE latency after model establishment (P<0.05), a significantly lower Lado score of seizure (P<0.05), significantly lower frequency and amplitude of epileptiform discharges on EEG (P<0.05), and a slightly reduced mortality rate.

CONCLUSIONSThe treatment with the microRNA-132 antagonist shows an inhibitory effect on the development and progression of lithium-pilocarpine-induced SE in young SD rats. The inhibition of microRNA-132 is likely to be a potential target or direction for drug treatment of SE.

Animals ; Electroencephalography ; Male ; MicroRNAs ; antagonists & inhibitors ; Pilocarpine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Status Epilepticus ; chemically induced ; drug therapy

OBJECTIVETo investigate the effect of licorice flavonoid (LF) on kainic acid (KA)-induced seizure in mice and its mechanism.

METHODSMale adult ICR mice were injected with 25 mg/kg KA to induce temporal lobe seizure. LF was administrated 7 d before seizure induction (pre-treatment) or 24 h after seizure induction (post-treatment) for 7 d. Acute seizure latency, seizure stage and duration were observed and compared between LF- and vehicle-treated mice. From d2 on, mice with status epilepticus were video-monitored for spontaneous seizures, 10 h/d for 6 w. Immunohistochemical analysis of BrdU and Timm staining was conducted to detect the neurogenesis and mossy fiber sprouting, respectively.

RESULTSNo significant difference was observed in acute seizure latency, seizure stage and duration between LF-and vehicle-treated mice. KA-induced acute seizure resulted in spontaneous seizure in mice, and the seizure frequency was increased with time. Pre- and post-treatment with LF decreased seizure frequency from w3 after modeling [(0.58±0.15)/d, (0.38±0.38)/d vs (1.23±0.23)/d, P <0.05]. Furthermore, KA-induced seizure resulted in robust neurogenesis and mossy fiber sprouting, while treatment with LF both pre- and post- KA injection significantly inhibited neurogenesis (15.6±2.6, 17.1±3.1 vs 28.9±3.5, P <0.05) and mossy fiber sprouting (1.33±0.31, 1.56±0.42 vs 3.0±0.37, P <0.05).

CONCLUSIONLF has no significant anti-seizure effect. However, it can decrease epileptogenesis through inhibition of neurogenesis and mossy fiber sprouting.

Animals ; Disease Models, Animal ; Flavonoids ; pharmacology ; Glycyrrhiza ; chemistry ; Kainic Acid ; adverse effects ; Male ; Mice ; Mice, Inbred ICR ; Mossy Fibers, Hippocampal ; drug effects ; Neurogenesis ; drug effects ; Seizures ; chemically induced ; drug therapy ; Status Epilepticus ; drug therapy

The present study is to explore the change process and distribution of phosphorylated DARPP-32 (p-DARPP-32) in rat brain including cortex, hippocampus and striatum and to further deduce whether p-DARPP-32 was possibly involved in epilepsy induced by repetitive low doses of pentylenetetrazol (PTZ). PTZ-induced epilepsy model in rat was established with 30 male SD rats randomly divided into 6 groups, control group and five trial groups [PTZ 1 h, PTZ 6 h, PTZ 24 h, PTZ 48 h and PTZ 72 h respectively, after onset of status epilepticus (SE)]. Immunohistochemistry and immunofluorescence double-labeling were used to detect the temporal time change and distribution of p-DARPP-32 expression and to analyze the coexpression of DARPP-32 and p-DARPP-32 in rat brain after the onset of PTZ-induced generalized SE. The results showed that there was a temporal time change of p-DARPP-32 expression in rat brain after the onset of SE. The number of p-DARPP-32-positive cells increased significantly and reached the peaks at the ends of 1 hour and 6 hours after the onset of SE, but decreased at the end of 24 hours. The moderate to strong p-DARPP-32-immunopositive neurons were observed in cortex, hippocampus and striatum, and located in cell cytoplasm and cell nucleus. Further immunofluorescence double-labeling revealed that denser colocalization of p-DARPP-32 and DARPP-32 in the neurons existed in the area mentioned above. Therefore, PTZ-induced SE may cause phosphorylation of DARPP-32 in rat brain. The temporal time change and distribution of p-DARPP-32 suggest that phosphorylation of DARPP-32 may be involved in PTZ-induced epilepsy in rat brain including cortex, hippocampus and striatum, and p-DARPP-32 may play a central role in the onset of SE.
Animals ; Cerebral Cortex ; metabolism ; Corpus Striatum ; metabolism ; Dopamine and cAMP-Regulated Phosphoprotein 32 ; metabolism ; Hippocampus ; metabolism ; Male ; Neurons ; metabolism ; Pentylenetetrazole ; Rats ; Rats, Sprague-Dawley ; Status Epilepticus ; chemically induced ; metabolism

BACKGROUNDAnnexin A7 (synexin, ANXA7) is a member of annexins, which plays an essential role in the regulation of calcium homeostasis. Considerable evidence shows that the pathogenetic mechanism of acquired epilepsy (AE) has been related to the imbalance of calcium homeostasis. The aim of this study was to investigate ANXA7 expression and cellular localization in the cortex and hippocampus in the rat lithium-pilocarpine model of AE.

METHODSTotally 81 adult healthy male Wistar rats were randomly divided into control group (n = 9) and experimental group (n = 72), the experimental group contained eight subgroups according to sacrifice time (n = 9) (6-hour, 24-hour, 48-hour, 72-hour, 7-day, 15-day, 1-month, and 2-month). In the experimental group, rats were intraperitoneally injected by lithium-pilocarpine to induce AE model. We examined the expression and localization of ANXA7 via immunohistochemistry, double-label immunofluorescence with the use of neuron specific enolase (NSE) antibody, glial fibrillary acidic protein (GFAP) antibody and propidium iodide (PI), respectively. The data of optical density value were analyzed by analysis of variance.

RESULTSANXA7 expression increased significantly in the experimental groups especially in the acute period (6 hours, 24 hours, and 48 hours after the onset of seizure) using immunohistochemistry. Double-label immunofluorescence and confocal microscopy disclosed that ANXA7 localized in the neurons but not in astrocytes and did not localize in the nucleus, which were performed with anti-NSE, anti-GFAP and PI respectively.

CONCLUSIONANXA7 may play a potential role in the pathogenetic mechanisms of the rat lithium-pilocarpine model of AE.

Animals ; Annexin A7 ; analysis ; physiology ; Calcium ; metabolism ; Cerebral Cortex ; chemistry ; Disease Models, Animal ; Fluorescent Antibody Technique ; Hippocampus ; chemistry ; Immunohistochemistry ; Lithium Chloride ; Male ; Pilocarpine ; Rats ; Rats, Wistar ; Status Epilepticus ; chemically induced ; metabolism

PURPOSE: Lithium-pilocarpine induced status epilepticus (LPSE) causes selective and age-dependent neuronal death, although the mechanism of maturation-related injury has not yet been clarified. The activating transcription factor-2 (ATF-2) protein is essential for the normal development of mammalian brain and is activated by c-Jun N-terminal kinase (JNK). It induces the expression of the c-jun gene and modulates the function of the c-Jun protein, a mediator of neuronal death and survival. Therefore, we investigated the expression of c-Jun and ATF-2 protein in the immature and adult rat hippocampus to understand their roles in LPSE-induced neuronal death. MATERIALS AND METHODS: Lithium chloride was administrated to P10 and adult rats followed by pilocarpine. Neuronal injury was assessed by silver and cresyl violet staining, performed 72 hours after status epilepticus. For evaluation of the expression of ATF-2 and c-Jun by immunohistochemical method and Western blot, animals were sacrificed at 0, 4, 24, and 72 hours after the initiation of seizure. RESULTS: Neuronal injury and expression of c-Jun were maturation-dependently increased by LPSE, whereas ATF-2 immunoreactivity decreased in the mature brain. Since both c-Jun and ATF-2 are activated by JNK, and targets and competitors in the same signal transduction cascade, we could speculate that ATF-2 may compete with c-Jun for JNK phosphorylation. CONCLUSION: The results suggested a neuroprotective role of ATF-2 in this maturation-related evolution of neuronal cell death from status epilepticus.
Activating Transcription Factor 2/*metabolism ; Animals ; Antimanic Agents/pharmacology ; Blotting, Western ; Hippocampus/drug effects/*metabolism ; Immunohistochemistry ; Lithium/pharmacology ; Miotics/pharmacology ; Pilocarpine/pharmacology ; Proto-Oncogene Proteins c-jun/*metabolism ; Rats ; Status Epilepticus/*chemically induced

OBJECTIVE: To investigate the role of neuropeptide Y(NPY) positive interneurons in the generation and compensation of temporal lobe epilepsy. METHODS: Pilocarpine-induced rat model was founded. Immunohistochemistry was used to observe the number changes and axonal sprouting of NPY interneurons at different time points in the hippocampus of rats. RESULTS: After lithium-chloride and pilocarpine administration, 92.9% rats were induced status epilepticus (SE) successfully, and the mortality rate was 19.2%. In the experimental group, the number of NPY positive neurons decreased in the hilus of the hippocampus, and was least on 7 d after the SE (P<0.01). In the chronic phase, the number of hilus NPY neurons partially recovered, but was still less than the number in the control group on 60 d after the SE (P<0.05). No evident changes of the number of NPY neurons existed in CA domains (P>0.05) except the loss of them in CA3 area on 7 d after the SE (P>0.05). Increased NPY positive fibers could be seen in the molecular layer of the dentate gyrus on 30 d after the SE. CONCLUSION NPY interneurons have different sensitivities to the injuries induced by seizures at different time points and domains. Loss of NPY interneurons plays an important role in the generation of temporal lobe epilepsy, while axonal sprouting of them may play a significant role in the compensation of temporal lobe epilepsy.
Animals ; Epilepsy, Temporal Lobe ; chemically induced ; pathology ; Hippocampus ; metabolism ; pathology ; Interneurons ; metabolism ; pathology ; Male ; Neuropeptide Y ; metabolism ; Pilocarpine ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Retrograde Degeneration ; pathology ; Status Epilepticus ; chemically induced ; pathology

OBJECTIVETo observe the changes in the learning and memory functions and the hippocampal expression of phosphorylated cAMP response element-binding protein (pCREB) in rats with status epilepticus and generalized nonconvulsive status epilepticus.

METHODSStatus epilepticus (SE) and generalized nonconvulsive status epilepticus (GNCSE) was induced by pentylenetetrazol kindling in SD rats, and the learning and memory function changes of the kindled rats were assessed by means of Morris water-maze test and Y-maze test with alternative electric stimulation. Immunocytochemistry was used for analysis pCREB protein expression in the hippocampus of the rats.

RESULTSIn Morris water-maze test, the rats with SE showed prolonged mean escape latency (P<0.05), shortened swimming time in the platform quadrant (P<0.05), and reduced number of times of platform crossing (P<0.05) in the short term after kindling. But these changes were reversed and became normal a month after the kindling (P>0.05). In the Y-maze test with alternative electric stimulation, the total error (TE) of SE rats increased significantly in the short term after epilepsy (P<0.05), but recovered the normal level a month after kindling (P>0.05). The GNCSE rats showed prolonged mean escape latency at only certain time periods (P<0.05) in the short term, but with swimming time in the platform quadrant and number of platform crossings similar to the control group (P>0.05). The short-term TE of GNCSE rats increased significantly (P<0.05), but in the long term, TE was similar to that in the control group (P>0.05). The expression of pCREB decreased significantly in SE group in comparison with the control group in the short term.

CONCLUSIONEpileptic seizures can lead to learning and memory function impairment in rats, and SE seems to cause greater impact than GNCSE on the learning and memory functions. pCREB might be involved in the pathophysiology of learning and memory deficit in epileptic rats.

Animals ; CREB-Binding Protein ; metabolism ; Hippocampus ; metabolism ; physiopathology ; Kindling, Neurologic ; Maze Learning ; Memory Disorders ; physiopathology ; Pentylenetetrazole ; Rats ; Rats, Sprague-Dawley ; Status Epilepticus ; chemically induced ; metabolism ; physiopathology

OBJECTIVETo examine modulations caused by cyclooxygenase-2 (COX-2) inhibitors on altered microenvironments and overbalanced neurotransmitters in pilocarpine-induced epileptic status rats and to investigate possible mechanisms.

METHODSCelecoxib (a COX-2 inhibitor) was administered 45 min prior to pilocarpine administration. The effects of COX-2 inhibitors on mIPSCs (miniature GABAergic inhibitory postsynaptic currents) of CA3 pyramidal cells in the hippocampus were recorded. Expressions of COX-2, c-Fos, newly generated neurons, and activated microgliosis were analyzed by immunohistochemistry, and expressions of alpha-subunit of gamma-amino butyric acid (GABA(A)) receptors and mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) activity were detected by Western blotting.

RESULTSPretreatment with celecoxib showed protection against pilocarpine-induced seizures. Celecoxib prevented microglia activation in the hilus and inhibited the abnormal neurogenesis and astrogliosis in the hippocampus by inhibiting MAPK/ERK activity and c-Fos transcription. Celecoxib also up-regulated the expression of GABA(A) receptors. NS-398 (N-2-cyclohexyloxy-4-nitrophenyl-methanesulfonamide), another COX-2 inhibitor, enhanced the frequency and decay time of mIPSCs.

CONCLUSIONThe COX-2 inhibitor celecoxib decreased neuronal excitability and prevented epileptogenesis in pilocarpine-induced status epilepticus rats. Celecoxib regulates synaptic reorganization by inhibiting astrogliosis and ectopic neurogenesis by attenuating MAPK/ERK signal activity, mediated by a GABAergic mechanism.

Animals ; Blotting, Western ; Celecoxib ; Cyclooxygenase 2 ; metabolism ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Disease Models, Animal ; Fibrocystic Breast Disease ; metabolism ; Hippocampus ; drug effects ; enzymology ; pathology ; Immunohistochemistry ; MAP Kinase Signaling System ; drug effects ; Male ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Nitrobenzenes ; pharmacology ; Pilocarpine ; Proto-Oncogene Proteins c-fos ; metabolism ; Pyrazoles ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; biosynthesis ; Status Epilepticus ; chemically induced ; enzymology ; pathology ; Sulfonamides ; pharmacology ; Synapses ; drug effects ; pathology

Adult ; Bed Rest ; adverse effects ; Coma ; chemically induced ; complications ; Encephalitis ; complications ; Female ; Humans ; Ossification, Heterotopic ; etiology ; Status Epilepticus ; complications ; drug therapy