Involvement of TRPV4 in changes in rapidly inactivating potassium channels in the early stage of pilocarpine-induced status epilepticus in mice.

The rapidly inactivating potassium current (IA ) is important in controlling neuronal action potentials. Altered IA function and K+ channel expression have been found in epilepsy, and activation of the transient receptor potential vanilloid 4 (TRPV4) channel is involved in epilepsy pathogenesis. This study examined whether TRPV4 affects Kv4.2 and K+ channel interacting protein (KCHIP) expression and IA changes following pilocarpine-induced status epilepticus (PISE) in mice. Herein, hippocampal protein levels of Kv4.2 and KCHIP2 increased 3 h-3 d and decreased 7-30 d; that of KCHIP1 increased 3-24 h and decreased 3-30 d post-PISE. The TRPV4 antagonist HC-067047 attenuated the increased protein levels of Kv4.2 and KCHIP2 but not that of KCHIP1 post-PISE. The TRPV4 agonist GSK1016790A increased hippocampal protein levels of Kv4.2 and KCHIP2 but had no effect on KCHIP1 expression. HC-067047 attenuated the increased IA in hippocampal pyramidal neurons 24 h and 3 d post-PISE. GSK1016790A increased IA in hippocampal pyramidal neurons, shifting the voltage-dependent inactivation curve toward depolarization. The GSK1016790A-induced increase of IA was blocked by protein kinase A and calcium/calmodulin-dependent kinase II antagonists but was unaffected by protein kinase C antagonists. We conclude that TRPV4 activation may be responsible for the increases of Kv4.2 and KCHIP2 expression in hippocampi and IA in hippocampal pyramidal neurons in PISE mice, which are likely compensatory measures for hyperexcitability at the early stage of epilepsy.

Transient receptor potential vanilloid 4 activation inhibits the delayed rectifier potassium channels in hippocampal pyramidal neurons: An implication in pathological changes following pilocarpine-induced status epilepticus.

Activation of transient receptor potential vanilloid 4 (TRPV4) can increase hippocampal neuronal excitability. TRPV4 has been reported to be involved in the pathogenesis of epilepsy. Voltage-gated potassium channels (VGPCs) play an important role in regulating neuronal excitability and abnormal VGPCs expression or function is related to epilepsy. Here, we examined the effect of TRPV4 activation on the delayed rectifier potassium current (IK ) in hippocampal pyramidal neurons and on the Kv subunits expression in male mice. We also explored the role of TRPV4 in changes in Kv subunits expression in male mice following pilocarpine-induced status epilepticus (PISE). Application of TRPV4 agonists, GSK1016790A and 5,6-EET, markedly reduced IK in hippocampal pyramidal neurons and shifted the voltage-dependent inactivation curve to the hyperpolarizing direction. GSK1016790A- and 5,6-EET-induced inhibition of IK was blocked by TRPV4 specific antagonists, HC-067047 and RN1734. GSK1016790A-induced inhibition of IK was markedly attenuated by calcium/calmodulin-dependent kinase II (CaMKII) antagonist. Application of GSK1016790A for up to 1 hr did not change the hippocampal protein levels of Kv1.1, Kv1.2, or Kv2.1. Intracerebroventricular injection of GSK1016790A for 3 d reduced the hippocampal protein levels of Kv1.2 and Kv2.1, leaving that of Kv1.1 unchanged. Kv1.2 and Kv2.1 protein levels as well as IK reduced markedly in hippocampi on day 3 post PISE, which was significantly reversed by HC-067047. We conclude that activation of TRPV4 inhibits IK in hippocampal pyramidal neurons, possibly by activating CaMKII. TRPV4-induced decrease in Kv1.2 and Kv2.1 expression and IK may be involved in the pathological changes following PISE.

Loss of Protection by Antiepileptic Drugs in Lipopolysaccharide-primed Pilocarpine-induced Status Epilepticus is Mediated via Inflammatory Signalling.

The evidences from various studies show the association of peripheral and neuronal inflammation with complex pathophysiology of status epilepticus (SE). In this view, the present work attempted to develop a model of neuronal inflammation mediated SE by combining both epileptic and inflammatory components of the disease and also to mimic SE co-morbid with systemic inflammation by peripheral administration of the lipopolysaccharide (LPS) 2 h prior to the pilocarpine (PILO) induction in C57BL/6 mice. We evaluated the anti-convulsant and neuroprotective effects of 7-day prophylactic treatment with three conventional anti-epileptic drugs (Sodium valproate, SVP 300 mg/kg p.o.; Carbamazepine CBZ 100 mg/kg p.o.; Levetiracetam; LEV 200 mg/kg p.o.) of widespread clinical use. Morris water maze and Rota rod tests were carried out 24-h post-exposure to evaluate the neurobehavioral co-morbidities associated with neuroinflammation-mediated status epilepticus. Upon priming with LPS, the loss of protection against PILO-induced seizures was observed by SVP and CBZ, however, LEV showed protection by delaying the seizures. Dramatic elevation in the seizure severity and neuronal loss demonstrated the possible pro-convulsant effect of LPS in the PILO model. Also, the decreased cytokine levels by the AEDs showed their association with NF-κB, IL-1ß, IL-6, TNF-α and TGF-ß pathways in PILO model. The loss of protective activities of SVP and CBZ in LPS+PILO model was due to increased cytokine levels associated with over-activation of neuroinflammatory pathways, however, partial efficacy of LEV is possibly due to association of other neuroinflammatory mechanisms. The current work provides direct evidence of the contribution of increased peripheral and neuronal inflammation in seizures via regulation of inflammatory pathways in the brain.

Decreased neuron loss and memory dysfunction in pilocarpine-treated rats pre-exposed to hypoxia.

Preconditioning can induce a cascade of cellular events leading to neuroprotection against subsequent brain insults. In this study, we investigated the chronic effects of hypoxic preconditioning on spontaneous recurrent seizures (SRS), neuronal death, and spatial memory performance in rats subjected to pilocarpine (Pilo)-induced status epilepticus (SE). Rats underwent a short hypoxic episode (7% O2+93% N2; 30min on two consecutive days) preceding a 4-h SE (HSE group). Control groups were rats submitted to SE only (SE), rats subjected to hypoxia only (H) or normoxia-saline (C). Animals were monitored for the occurrence of SRS, and spatial memory performance was evaluated in the radial-arm maze. Hippocampal sections were analyzed for cell death and mossy fiber sprouting at 1 or 60days after SE. Compared to SE group, HSE had increased SE latency, reduced number of rats with SRS, reduced mossy fiber sprouting at 60days, and reduced cell death in the hilus and the CA3 region 1 and 60days after SE. Additionally, HSE rats had better spatial memory performance than SE rats. Our findings indicated that short hypoxic preconditioning preceding SE promotes long-lasting protective effects on neuron survival and spatial memory.

Pyrrolidine dithiocarbamate (PDTC) inhibits the overexpression of MCP-1 and attenuates microglial activation in the hippocampus of a pilocarpine-induced status epilepticus rat model.

The aim of this study was to investigate the effects of pyrrolidine dithiocarbamate (PDTC) on MCP-1 expression and microglial activation in the hippocampus of a rat model of pilocarpine (PILO)-induced status epilepticus (SE). Moreover, seizure susceptibility, frequency and severity as well as brain damage were analyzed and changes in behavior were recorded. Chemokine MCP-1 expression and microglial activation were detected by immunohistochemistry (IHC). Fluoro-Jade C (FJC) and NeuN staining were used for the evaluation of tissue damage. Our results showed that although SE resulted in the upregulation of MCP-1 and microglial activation in the rat hippocampus 24 h after seizure onset, pretreatment with PDTC significantly inhibited the MCP-1 overexpression and attenuated the microglial activation. These effects were accompanied by neurodegenerative amelioration. To the best of our knowledge, these findings indicated for the first time that the activation of the nuclear factor-κB (NF-κB) pathway may contribute to MCP-1 upregulation and microglial activation in the context of epilepsy. PDTC was also shown to exert anticonvulsant activity and to have a neuroprotective effect on the hippocampal CA1 and CA3 regions, potentially through attenuating microglial activation.

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.

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.

Nonlinear Dynamics of EEG in Pilocarpine-induced Status Epilepticus

BACKGROUND: Pilocarpine-induced status epilepticus (SE) model shows stereotypic EEG changes and behavioral characteristics. Although neuronal damages and therapeutic responses are also dependent on SE stages, the dynamical aspect of underlying neuronal interaction according to the SE stages has not been studied. We applied nonlinear analysis to the EEG recorded from pilocarpine-induced SE model to characterize nonlinear dynamics of different SE stage and to correlate therapeutic response with correlation dimension (D2). METHODS: Ten male Spraque-Dawley rats weighing 150-250g were used in this experiment. EEG was continuously recorded during SE and was classified into 6 stages as follows: baseline (BS), transitional (TR), discrete seizure (DS), continuous ictal discharges (CID), early periodic epileptiform discharges (EP), and late periodic epileptiform discharges (LP). High dose diazepam (20 mg/kg) was injected at the LP stage. SEs of 5 rats (control group) was controlled by diazepam and those of the rest (failed group) were failed to stop status epilepticus. RESULTS: Mean D2 value decreased progressively with fluctuation and was significantly different for SE stage (df=5, F=11.594, p=0.000). Independent t-test showed that the difference of D2 value between the controlled and failed group was significant at CID (df=40, t=2.591, p=0.013) and LP stage (df=49, t=-2.425, p=0.019). CONCLUSIONS: These results suggest that nonlinear dynamical change at the late half of SE stage is one of the contributing factors determining therapeutic responsiveness in pilocarpine-induced SE model.

Nonlinear Dynamics of EEG in Pilocarpine-induced Status Epilepticus

BACKGROUND: Pilocarpine-induced status epilepticus (SE) model shows stereotypic EEG changes and behavioral characteristics. Although neuronal damages and therapeutic responses are also dependent on SE stages, the dynamical aspect of underlying neuronal interaction according to the SE stages has not been studied. We applied nonlinear analysis to the EEG recorded from pilocarpine-induced SE model to characterize nonlinear dynamics of different SE stage and to correlate therapeutic response with correlation dimension (D2). METHODS: Ten male Spraque-Dawley rats weighing 150-250g were used in this experiment. EEG was continuously recorded during SE and was classified into 6 stages as follows: baseline (BS), transitional (TR), discrete seizure (DS), continuous ictal discharges (CID), early periodic epileptiform discharges (EP), and late periodic epileptiform discharges (LP). High dose diazepam (20 mg/kg) was injected at the LP stage. SEs of 5 rats (control group) was controlled by diazepam and those of the rest (failed group) were failed to stop status epilepticus. RESULTS: Mean D2 value decreased progressively with fluctuation and was significantly different for SE stage (df=5, F=11.594, p=0.000). Independent t-test showed that the difference of D2 value between the controlled and failed group was significant at CID (df=40, t=2.591, p=0.013) and LP stage (df=49, t=-2.425, p=0.019). CONCLUSIONS: These results suggest that nonlinear dynamical change at the late half of SE stage is one of the contributing factors determining therapeutic responsiveness in pilocarpine-induced SE model.