Promotion of epileptic seizure through up-regulation of matrix metalloproteinase-9 by apoptosis signal-regulating kinase 1 in lithium-pilocarpine epilepsy model / 实用医学杂志

Objective To investigate the alteration of apoptosis signal-regulating kinase 1(ASK1)phos-phorylation and matrix metalloproteinase-9(MMP-9)expression after temporal lobe epilepsy(TLE),and to explore the role and mechanism of ASK1-MMP-9 signal in TLE. Methods The lithium-pilocarpine epilepsy model was established. The ASK1 phosphorylation and MMP-9 expression were detected by western blot and immunofluo-rescence histochemistry. Using ASK1 inhibitors,the regulating effects of ASK1 on MMP-9 expression were deter-mined. And the role of ASK1 and MMP-9 in epileptic seizure was explored by inhibition of ASK1 and MMP-9. Results The ASK1 phosphorylation and MMP-9 expression increased in hippocampus in epilepsy model ,exhibiting a temporal correlation with epileptic seizure. Inhibition of ASK1 activation reduced the expression of MMP-9. And the level of epileptic seizure was significantly reduced by the inhibition of ASK1 and MMP-9. Conclusions The hippocampal ASK1 phosphorylation and MMP-9 expression distinctly increase in TLE model ,and the MMP-9 expression is induced by ASK1 activation. The ASK1-MMP-9 signal plays an important role in TLE.

Preserved Hippocampal Glucose Metabolism on 18F-FDG PET after Transplantation of Human Umbilical Cord Blood-derived Mesenchymal Stem Cells in Chronic Epileptic Rats

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) may be a promising modality for treating medial temporal lobe epilepsy. 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) is a noninvasive method for monitoring in vivo glucose metabolism. We evaluated the efficacy of hUCB-MSCs transplantation in chronic epileptic rats using FDG-PET. Rats with recurrent seizures were randomly assigned into three groups: the stem cell treatment (SCT) group received hUCB-MSCs transplantation into the right hippocampus, the sham control (ShC) group received same procedure with saline, and the positive control (PC) group consisted of treatment-negative epileptic rats. Normal rats received hUCB-MSCs transplantation acted as the negative control (NC). FDG-PET was performed at pre-treatment baseline and 1- and 8-week posttreatment. Hippocampal volume was evaluated and histological examination was done. In the SCT group, bilateral hippocampi at 8-week after transplantation showed significantly higher glucose metabolism (0.990 +/- 0.032) than the ShC (0.873 +/- 0.087; P < 0.001) and PC groups (0.858 +/- 0.093; P < 0.001). Histological examination resulted that the transplanted hUCB-MSCs survived in the ipsilateral hippocampus and migrated to the contralateral hippocampus but did not differentiate. In spite of successful engraftment, seizure frequency among the groups was not significantly different. Transplanted hUCB-MSCs can engraft and migrate, thereby partially restoring bilateral hippocampal glucose metabolism. The results suggest encouraging effect of hUCB-MSCs on restoring epileptic networks.

Differential Expression of Activating Transcription Factor-2 and c-Jun in the Immature and Adult Rat Hippocampus Following Lithium-Pilocarpine Induced Status Epilepticus

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.

The Effect and the Mechanism of Action of High Frequency Electrical Stimulation of Subthalamic Nucleus on Status Epilepticus Induced by Lithium-Pilocarpine of Rat / 대한간질학회지

OBJECTIVES: Deep brain stimulation (DBS) of subthalamic nuclei (STN) is one of the current modalities of refractory epilepsy, but its exact mechanism and route of action have not been elucidated yet. We investigated the effect of STN stimulation on the development and propagation of seizures in the rats with lithium-pilocarpine induced status epilepticus in its functional anatomy. METHODS: Both pilocarpine injection and high frequency stimulation on STN (HFSSTN) were provided to rats (STN group, n=12), but pilocarpine injection with no stimulation was done on the sham group (n=8). The latency to first discrete ictal discharges and the latency to status epilepticus (SE) were analyzed and the electrical stimulation lasted for 30, 60, 90, 120 minutes after its first discrete spikes. After stimulation, the rats were immediately decapitated for immunohistochemistry and histologic examination. RESULTS: Both the latency to first discrete ictal discharges and the latency to the onset of SE were delayed in the STN group than in the sham group. The latency to the first SE was also more delayed in the STN group (42.7+/-7.9 min) than in the sham group (p<0.05). Remarkably, there was marked Fos immunoreactivity (FIR) on the reticular thalamic nuclei in the STN group, but not in the sham group. CONCLUSIONS: Increased FIR in the reticular thalamic nuclei during HFSSTN suggested that the facilitation of the inhibitory thalamic output prevented generalized motor seizure behavior. We assume that HFSSTN has a pivotal role in the suppression or progression to SE, but cannot prevent seizure onset.

Antiepileptic and Neuroprotective Effect of Ketamine in Lithium-Pilocarpine Induced Status Epilepticus Rat Model / 대한간질학회지

PURPOSE: To examine the putative seizure-protective properties of ketamine in lithium-pilocarpine induced status epilepticus (LPSE). METHODS: Lithium chloride followed 24 h later by pilocarpine was administered for seizure induction. Ketamine (40 mg/kg) or phenytoin (50 mg/kg) was injected intraperitoneally 10 min or 60 min after the onset of continuous ictal discharge. Then the seizure behavior and EEG were observed and histological changes were compared through Nissl stain at 72 hours. RESULTS: The antiepileptic effect of ketamine, injected during the early stages of LPSE (10 min after the onset of continuous ictal discharge), was comparable to that of phenytoin. Ketamine was more effective than phenytoin in decreasing spike frequency, when administered on the plateau of LPSE (injection 60 min after onset of continuous ictal discharge electrographically). Anticonvulsant action of ketamine was confirmed by a less neuronal injury in hippocampus compared with control rats injected with phenytoin. CONCLUSIONS: In prolonged status epilepticus rat model, ketamine was effective as an antiepileptic, but phenytoin was not. Ketamine was also neuroprotective on the neuronal injury in the hippocampus. These results suggest that ketamine might be useful as an antiepileptic drug when standard antiepileptic drugs fail in the treatment of the refractory cases of status epilepticus.

The Effect of Corpus Callosotomy in the Lithium-Pilocarpine Induced Status Epileptic Rats

Section of the corpus callosum(SCC) is a useful surgical therapy in selected types of epilepsy, i.e., tonic, atonic, and intractable generalized convulsive seizures. The object of this study was to determine effect of SCC on behaviors, electroencephalography(EEG) and Fos expression in the lithium-pilocarpine model of status epilepticus in the rat. A total of 40 Sprague-Dawley rats were used. They were divided into two groups: control and lesioned group, 20 rats for each. The control group had no callosal section and was injected with lithium-pilocarpine. The lesioned group had callosal section before lithium-pilocarpine injection. In each group, ten rats were used for behavior and EEG monitoring and other 10 were used for Fos expression. The results were as follows: 1) In the SCC group, four(40%) rats never developed status epilepticus, among them two(20%) never exhibited any seizure, while all of the control group developed seizure and status epilepticus. None of the SCC animals died until 24 hours after lithium-pilocarpine injection but 70% of the control animals died within 24 hours of status epilepticus. This difference was statistically significant(p<0.05). 2) The mean latency to the first seizure, status epilepticus and periodic epileptiform discharges after lithium-pilocarpine injection were 34.7+/-2.6min, 32.3+/-1.8min and 180.4+/-9.8min, respectively, in the SCC group, while was 21.0+/-2.0min, 58.2+/-6.9min and 215.6+/-7.2min, respectively, in the control group. These latencies were significantly longer than in the control group(p<0.05). 3) There was a massive Fos expression on the cerebral cortex in the control group at 4 hours after lithiumpi-locarpine injection , while it was less in the SCC group. This difference was statistically significant(p<0.05). In conclusion, complete corpus callosotomy had contributed to the protective effect on the development of status epilepticus in the lithium-pilocarpine model which was similar to that observed in humans. And result of Fos expression suggest that Fos immunohistochemisty may be useful in the study of seizure pathways as a metabolic marker in the lithium-pilocarpine model.

A Preliminary Study for the Estimation of the First Positive Lyapunov Exponent of the EEG in Experimental Status Epilepticus Rats / 대한간질학회지

BACKGROUND AND OBJECTIVES: The hypothesis that brain is a nonlinear dynamic system exhibiting deterministic chaos has offered new methods to the investigation of information processing in the brain by analysis and classification of EEG signals. We used the first positiveLyapunov exponent (L1) which is one of indicator of nonlinear dynamic to evaluate the brain function in chemical seizure models METHODS: Lithium-Pilocarpine induced seizure model and kainic acid induced seizure model are used. From serial EEG according to seizure stages. 32.768 sec of data (16.384 data point) were recorded and digirized by a 12-bit analog-digital converter in an IBM PC. The data from serial EEG according to seizure stageswere analyzed for determining the L1. We used the time delays calculated by the method of mutual information to reconstruct the attactor. Time delays of 46-58 msec and enbedding dimensions of 13-19 were used for chemical seizure model. The L1 were calculated for 4 channels. RESULTS: The averaged valued of L1 were serially decreased in both lithium-pilocarpin model and kainic acid model according to increasing seizure stages. CONCLUSION: Our results reveal the decrease of the chaotic activity according to increasing seizure stage. It is suggested that the brain has decreased information procedding and a less flexible neural network during seizure. However further evaluation is required because the significance of these changes are not confirmed.

Effect of MK-801 on Neuronal Cell Loss and Fos Expression of Hippocampus in Lithium-Pilocarpine Induced Status Epilepticus / 대한간질학회지

BACKGROUND & OBJECTIVES: Endogenous excitatory amino acid has been implicated in neuronal damages occurred neurotransmitter in the CNS, Exerts its neurotoxic activity primarily by binding to the NMDA receptor, one of the three glutamate receptors, We evaluated the effect of MK-801, a non-competitive NMDA receptor antagonist, on both Fos expression and neuronal damages in lithium-pilocarpine induced status epilepticus rat model. METHODS: Seizured was induced in rats by lithum pretreatment followed by low dose of pilocarpine (30 mg/kg, ip). MK-801 (1 mg/kg) was treated 15 min before (MK-801 Pre group, N=5) or 20 min after the injection of pilocarpine (MK-801 Post group, N-5). Saline, instead of MK-801, was injected for the Control group (N=5). RESULTS: Seizure-induced neuronal damages, which was evaluated by the counting of the number of viable pyramidal cells in the area of CA1 and CA3 of the hippocampus, were significantly inhibited by MK-801 in both MK-801 Pre and Post groups. This protective effect of MK-801 was observed only in the CA1 area and was not typical in the CA3 area in both groups, and there was no differences in MK-801 activity between Pre and Post group. We also examined the expression of Fos, which has been known to be involved in long-term neuronal plasticty or delayed neuronal insults, by the immunohistochemical analysis in the hippocampus and thalamus. Pilocarpine induced Fos expression significantly in the Control group and moderately in the MK-801 Post groups, whereas, it was completely blocked by the pretreatment of MK-801 in hippocampus. CONCLUSION: Our results showed that MK-801 prevented the hippicampal cells from neuronal damages as well as inhibited Fos expression in the pilocarpine-induced rat seizure models. These results suggest the roles of NMDA receptor and Fos protein in seizure-related hippicampal damages.

Effect of Body Temperature on Seizure and Hippocampal Damage in Rats with Lithium-Pilocarpine Induced Status Epilepticus / 대한간질학회지

BACKGROUND: To investigate the effect of body temperature on seizure and hippocampal damage in the lithium and pilocarpine-induced status epilepticus METHODS: We pretreated the Sprague-Dawley rats with lithium (3mEq/kg) and pilocapine (30 mg/kg) and divided into three groups by their body temperatures which were maintained at normothermia (36.5+/-0.5 degrees C), hyperthermia(40.0+/-0.5 degrees C) and hypothermia (320+/-0.5 degrees C). Each group was maintained in a state of status epilepticus with assigned body temperatyre for 4 hours. After 24 hours, the rats were sacrificed, the pattern of EEG changes and degree of hippocampal cellular damage were compared between different groups. RESULTS: (1) Progression of typical EEG pattern of status epilepticus were observed in normothermic group ; discrete ictal discharge with slowing (30 min), wax and waning ictal discharge (60 min), continuous ictal discharge (90 min), continuous ictal discharge with flat period (180 min) and periodic epileptiform discharge (240 min) in time orfer. In hyperthermic group, the progression of EEG pattern was more rapid tha that od normothermic group ; continuos ictal dischatge was appeared within 30 minutes and periodic epileptiform discharge was within 90 minutes. On the other hand, the hypothermic group showed delayed evolution and wax and waning ictal discharge persisted until 240 minutes. (2) The survived neuronal cellular counts at CA1 and CA3 area of ventral hippocampus were compared between each group. The survived neuronal cellular counts were 90.9+/-5.55 (CA1) and 73.9+/-5.15 (CA3) in hypithermic group, 56.1+/-5.38 (CA1) and 40.6+/-7.03 (CA3) in normothermic group and 42.7+/-4.08 (CA1) and 31.7+/-4.64 (CA3) in hyperthermic group (p<0.05). CONCLUSION: The body temperatyre might influence the severity of seizure and seizure-induced brain damage. Hyperthermia aggravated the severity of seizure and seizure induced hippocampal damage, while hypothermia inhibited them.

Effect of electroacupuncture applied at acupoint on cognitive function in lithium-pilocarpine induced spontaneous recurrent epileptic rat / 西安交通大学学报(医学版)

Objective To investigate the effect of electroacupuncture applied at Baihui acupoint on cognitive function in lithium-pilocarpine induced spontaneous recurrent epileptic rat and provide some theoretical basis for acupoint stimulation treatment seizure.Methods Male mature Sprague Dawley rats were subjected to spontaneous recurrent epileptic model induced by lithium-pilocarpine,and divided into different groups randomly: acupoint group(electroacupuncture applied at Baihui acupoint),non-acupoint group(electroacupuncture located in close vicinity to the Baihui acupoint),and epileptic control group.Normal rats were subjected into normal control group.The Morris water maze test and step though test were carried out respectively to explore the effect of electroacupuncture applied at Baihui acupoint on the rats' learning and memory capacities.Results Compared with control group,acupoint rats in Morris water maze test time of finding the platform under the water surface decreased(P