| Part one The expression pattern of MyD88 in patients with refractory epilepsy and epileptic ratsObjective:Accumulating evidence supports that activation of inflammatory pathways is a common factor contributing to the pathogenesis of seizures. Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein, which plays a crucial role in immune and inflammatory responses. The present study aimed to investigate the expression pattern of MyD88 in patients with refractory temporal lobe epilepsy (TLE) and in different rat models of seizures.Methods:1. The temporal neocortex of twenty-five patients undergoing surgery for medically intractable TLE and fifteen nonepileptic control subjects were chosen randomly from our established brain tissue bank. MyD88 protein expression was determined through double-immunofluorescence labelling, immunohistochemistry, and western blotting.2. The expression pattern of MyD88 were examined in the rat hippocampus and the adjacent cortex at 6,24, and 72 h following pilocarpine (25mg/kg)-induced acute seizures through western blotting and double-immunofluorescence labelling. In the aucte PTZ model, MyD88 protein was examined at 3,6, and 24 h following PTZ injection (65 mg/kg) by western blotting.3. The lithium-pilocarpine model of TLE was made as previously reported in our laboratory. At about 2 m after pilocarpine injection, rats with chronic spontaneous recurrent seizures (SRS) were sacrificed to analyze MyD88 expression through immunohistochemistry and western blotting. In the chronic PTZ kindling model, MyD88 protein level was determined by western blotting.Results:1. MyD88 was significantly up-regulated in the temporal neocortex specimens from drug-resistant TLE patients. MyD88 was exclusively expressed in the cytoplasm of neurons.2. MyD88 expression was significantly increased at 24 h and 72 h after pilocarpine injection in the lithium-pilocarpine model of acute seizures and was expressed in neurons but not in astrocytes. In acute PTZ model, MyD88 expression was markedly increased at 6 h and 24 h after PTZ injection.3. MyD88 overexpression was also significantly present in chronic epilepitc rats with SRS in the pilocarpine model of TLE. MyD88 overexpression was also observed in kindled rats in the chronic PTZ kindling model.Conclusions:MyD88 expression was up-regulated in patients with drug-resistant TLE and in different rat models of seizures, indicating that epileptic activity could affect MyD88 protein expression.Part two Effect of MyD88 pharmacological intervention in epileptic seizures in ratsObjective:ST2825 is a small synthetic MyD88 inhibitor. IL-1β is a proinflammatory cytokine that could promote MyD88 function. To explore the role of MyD88 in the onset and recurrence of seizures, ST2825 and IL-1β effect on epileptic seizures was assessed in acute pilocarpine model and in acute PTZ model.Methods:1. In the acute pilocarpine model, ST2825 was injected intracerebroventricularly(i.c.v.) at doses of 2.5,5.0, and 10.0 μg 20 min before pilocarpine. The behavioral seizures were closely observed for 90 min after pilocarpine injection. The number of rats developing status epilepticus (SE), the latency to SE onset, the seizure severity score, and the accumulative seizure severity score were used as outcome measures.2. In the acute pilocarpine model,10 μg of ST2825 was administered i.c.v.20 min before pilocarpine, rats were EEG recorded for 90 min after pilocarpine injection to detect electrographic seizure activity. The latency to the first seizure, the total number of ictal episodes, and the total time spent in seizures were chosen to quantify EEG seizure activity. In addition, the effect of 10 μg of ST2825 on seizure-induced microglial activation was also examined.3. In the acute PTZ seizure model,10μg of ST2825 was administered i.c.v.20 min before PTZ. The behavioral seizures were closely observed for 1h after PTZ injection. The latency to first clonic seizure, the latency to generalized tonic-clonic seizure, and the time spent in generalized tonic-clonic seizure were used as outcome measures.4. In the acute pilocarpine model, to test the effect of IL-1β on epileptic seizures and ST2825 effect on IL-1β actions, ST2825 was administered i.c.v.20 min before pilocarpine, and 1ng of rat recombinant (rr) IL-1β was injected i.c.v.10 min before pilocarpine. Rats were allocated into three groups:the vehicle group, the IL-1β group, and the ST+IL-1β group.5. In the acute PTZ seizure model, to test the effect of IL-1β on epileptic seizures and ST2825 effect on IL-1β actions, ST2825 was administered i.c.v.20 min before PTZ, and 1ng of rat recombinant (rr) IL-1β was injected i.c.v.10 min before PTZ. Rats were allocated into three groups:the vehicle group, the IL-1β group, and the ST+IL-1β group.Results:1. In the acute pilocarpine model, treatment with ST2825 i.c.v. at doses of 5μg and 10 μg significantly delayed the time to SE onset, reduced the seizure severity score, and decreased the accumulative seizure severity score.2. In the acute pilocarpine model, the i.c.v. administration of 10 μg of ST2825 significantly delayed the time to the first EEG seizure onset, markedly reduced the total number of EEG seizures,and decreased the total time spent in EEG seizures. In addition,10 μg of ST2825 could suppress the seizure-induced microglial activation in the hippocampus and the adjacent cortex at 24 h following pilocarpine-induced seizures.3. In the acute PTZ model, the i.c.v. administration of 10 μg of ST2825 significantly delayed the latency to first clonic seizure, prolonged the latency to generalized tonic-clonic seizure, and shortened the time spent in generalized tonic-clonic seizures.4. In the acute pilocarpine model, the i.c.v. injection of 1 ng of IL-1β significantly shortened the time to SE onset and markedly exacerbated the seizure severity, and increased the accumulative seizure severity score.10 μg of ST2825 was administered 10 min before IL-1β. ST2825 pretreatment effectively prevented the IL-1β-induced reduction in the time to seizure onset, alleviated the IL-1β-induced exacerbation in seizure severity, and abolished the IL-1β-induced increase in accumulative seizure severity score. In addition, in terms of EEG seizure activity,1 ng of IL-1β significantly reduced the time to first EEG seizure onset and markedly prolonged the total time spent in EEG seizures. ST2825 pretreatment significantly prevented the IL-1β-induced reduction in latency to first EEG seizures, reduced the number of EEG seizures, and markedly blocked the IL-1β-induced increase in the total time spent in EEG seizures.5. In the acute PTZ model, the i.c.v. injection of 1 ng of IL-1β significantly shortened the latency to generalized tonic-clonic seizures and increased the time spent in generalized tonic-clonic seizures. Pretreatment with ST2825 markedly blocked the IL-1β-induced reduciton in seizure latency and the IL-1β-induced increase in time spent in generalized tonic-clonic seizures.Conclusions:1. MyD88 pharmacological inhibition with ST2825 suppressed behavioral and EEG seizure activities, and attenuated microglial activation in the acute pilocarpine model. In addtion, ST2825 inhibited seizures in acute PTZ model.2. IL-1β had proconvulsant actions in both acute pilocarpine model and acute PTZ model. ST2825 blocked the proconvulsant effects of IL-1β.Part three Potential mechanisms of MyD88 in epileptic seizures in ratsObjective:The tyrosine 1472-phosphorylation of the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor plays a critical role in neuronal hyperexcitability. Based on above studies, altered MyD88 function contributed to epileptic seizures, the present study examined whether the NR2B subunit of NMDA receptor was involved in the effect of the pharmacological treatments on seizures.Methods:1. The level of tyrosine phosphorylated forms of the NR2B subunit on Tyr1472 at 90 min after pilocarpine injection in the pharmacological treated rats was determined through western blotting. Rats were allocated into the following groups:the vehicle group, the Pilo group, the IL-1β group, the IL-1β+Pilo group, the ST+IL-1β+Pilo group, and the ST+Pilo group.2. In the acute pilocarpine model, to assess ifenprodil effect on IL-1β-seizure exacerbating actions, the NR2B inhibitor ifenprodil was administered i.c.v.20 min before pilocarpine, and 1ng of IL-1β was injected i.c.v.10 min before pilocarpine. Rats were allocated into four groups:the vehicle group, the IL-1(3 group, the Ifen group, and the Ifen+IL-1β group.3. In the acute PTZ model, to further assess ifenprodil effect on IL-1β-seizure exacerbating actions, the NR2B inhibitor ifenprodil was administered i.c.v.20 min before PTZ, and 1ng of IL-1β was injected i.c.v. 10 min before PTZ. Rats were allocated into four groups:the vehicle group, the IL-1β group, the Ifen group, and the Ifen+IL-1β group.Results:1. Either IL-1β alone (no seizures) or seizure per se increased the phosphorylated forms of the NR2B subunit on Tyr1472 by 154% and 134% on average, respectively (p<0.05). When IL-1β was administered 10 min before pilocarpine, thus producing proconvulsant effects, the phosphorylated NR2B levels was increased by 213% on average as compared to vehicle-treated rats (p<0.01), indicating addictive effects of the single treatments.10 μg of ST2825 pretreatment significantly prevented the increase in the NR2B phosphorylation in rats received pilocarpine alone (p<0.05). Moreover,10 μg of ST2825 treatment effectively blocked the increased NR2B phosphorylation in rats received IL-1β+pilocarpine (p<0 .01).2. In the acute pilocarpine model, the i.c.v. injection of 1 ng of IL-1β significantly shortened the time to SE onset and markedly exacerbated the seizure severity, and increased the accumulative seizure severity score. The NR2B Tyr1472 inhibitor ifenprodil effectively prevented the IL-1β-induced reduction in the time to seizure onset, alleviated the IL-1β-induced exacerbation in seizure severity, and abolished the IL-1β-induced increase in accumulative seizure severity score.3. In the acute PTZ model, compared with the vehicle group, IL-1β significantly shortened the latency to generalized tonic-clonic seizures and prolonged the time spent in generalized tonic-clonic seizures. In contrast, ifenprodil markedly delayed the latency to first clonic seizures and generalized tonic-clonic seizures, decreased the time spent in generalized tonic-clonic seizures, exerting an anticonvulsant effects. In addition, ifenprodil effectively blocked IL-1β-induced reduction in seizure latency and the IL-1β-induced increase in time spent in generalized tonic-clonic seizures.Conclusions:1. The phosphorylated forms of the NR2B subunit on Tyr1472 was increased in seizure conditions. IL-1β could further increased the Tyr1472-phosphorylated NR2B levels. By contrast, ST2825 pretreatment significantly prevented the increase in the NR2B Tyr1472-phosphorylation in rats.2. The NR2B subunit selective inhibitor ifenprodil could inhibit seizure acitivities, and might block the proconvulsant actions of IL-1β.Part four Effect of MyD88 pharmacological intervention on epileptogenensis in ratsObjective:Mounting evidence suggests that brain inflammation may contribute to epileptogenesis, and antiinflammatory therapy may represent a promising strategy to improve therapeutic effects on epilepsy. The present study further investigated the role of MyD88 in epileptogenesis.Methods:1. PTZ was administered intraperitoneally once daily at a dosage of 33 mg/kg rat body weight to establish chronic PTZ kindling model. Ing of rat recombinant (rr) IL-1β was injected i.c.v.10 min before PTZ. ST2825 was administered i.c.v.20 min before PTZ. Rats were allocated into the following groups:the Con group, the vehicle group, the IL-1β group, and the ST2825 group. The most severe seizure observed after each PTZ injection and the latency to fully kindling was recorded. The rats were considered to be kindled after exhibition of at least three consecutive seizures of class 4 or 5.2. In chronic pilocarpine epilepsy model, Both IL-1β and ST2825 was administered once daily for 7 days starting at 24h after termination of SE. SD rats were randomly allocated into the vehicle group, the IL-1β group, and the ST2825 group. SRS was determined by video monitoring from day 21 to day 34 after pilocarpine injection. SRS incidence, SRS frequency, and SRS severity were used as outcome measures and recorded for statistical analysis.Results:1. In the chronic PTZ kindling model, the latency to fully kindling was significantly shortened in the IL-1β group compared with the vehicle group. The seizrue severtiy score in the IL-1β group was significantly elevated at the 5,7-9,10,12 days compared with the vehicle group. In contrast, the latency to fully kindling was markedly prolonged in the ST2825 group compared with the vehicle group. The seizure severity score in the ST2825 group was markedly attenuated at the 6-8,13-18 days. In addtion, there were no significant differences in the latency to fully kindling and the seizure severity score between the vehicle group and the Con group.2. In the chronic pilocarpine model, there were no significant differences in SRS incidence among the vehicle group, the IL-1β group, and the ST2825 group. Compared with the vehicle group, the SRS frequency was markedly increased in the IL-1β group (p< 0.05), but was similar in the ST2825 group (p> 0.05). Compared with the vehicle group, the seizure severity was significantly increased in the IL-1β group (p< 0.05), but was not different from the ST2825 group (p> 0.05).Conclusions:1. In the chronic PTZ kindling model, IL-1β significantly shortened the latency to fully kinding and the kindling progress. By constrast, ST2825 significantly prolonged the time to fully kindling and delayed the kindling progress, indicating that ST2825 had some antiepileptogenic effects in the chronic PTZ kindling model.2. In the chronic pilocarpine epilepsy model, IL-1β could increase SRS frequency and exacerbate SRS severity. ST2825 had no effect on SRS frequency and severity. These results indicated that epileptogenesis induced by post-SE was more difficult to modify and may need combination of different approaches in future. |
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