The Role Of MiR134 In The Pathophysiologic Mechanism Of Status Epilepticus

Status epilepticus(SE) is the second most life-threatening neurologic emergencies after acute cerebrovascular disease. The annual incidence of SE in the world is estimated at 10-40 per 100, 000 per year. About one-third of SE patients fail to respond to two or more medications, and evolve into refractory SE, which often results in significant mortality and morbidity. Thus, it is urgent to explore new fast and effective treatments.Growing evidences show that micro RNAs(mi RNAs) play an important role in the mechanism of epileptogenesis. Mi RNAs are a family of small(19-24nt) endogenous highly conserved noncoding RNAs, which can degrade m RNA and suppress translation by specifically binding to one or more m RNA as the role of complementary base pairs, so mi RNAs negatively regulate the target gene at post-transcriptional level, which are involved in many pathophysiologic processes of nervous system. Mi R-134, specifically expressing in the nervous system, mainly participates in the modulation of neuron microstructure, such as the density and length of dendritic spines, which regulates the excitatory of local neural circuits. Several evidences suggest that mi R-134 in hippocampus is significantly upregulated in the process of SE. Combined with its specific expression and physiological function, we hypothesized that mi R-134 might be involved in the epiletptogenesis of SE. Intensive study of mi R-134 might provide a new therapeutic target for SE ObjectiveTo investigate the role of mi R134 in the pathophysiologic mechanism of status epilepticus Methods:1. Rat mi R-134 inhibited lentiviral vectors were constructed and verified for subsequent studies.2. Lithium chloride and pilocarpine were injected intraperitoneally in SD rats to establish SE model. The influence of knock down of mi R-134 by mi R-134 inhibited lentiviral vectors on the racine score Ⅴ lantency, duration of SE, severity, EEG power and neuron apoptosis in hippocampus were observed in vivo.3. The low-Mg2+ hippocampal neuronal culture model of SE-like electrographic activity was built. The influence of knock down of mi R-134 by mi R-134 inhibited lentiviral vectors on the neuronal discharge frequency and neuroprotection were observed.4. In vitro model of SE, the influence of knock down of mi R-134 by mi R-134 inhibited lentiviral vectors on the neuronal dendritic spines was observed.Results1. A synthesized 362 bp long DNA fragment which contain two mi R-134 competitive binding sites was inserted into downstream of EG(R)FP sequences in p CDH-CMV-MCS-EG(R)FP-MCS vector to get rat mi R-134 inhibitor lentiviral vector.The transcript of DNA fragments can specifically bind to mi R-134 to form a stable complex, which can inhibit the degradation and translation suppression of mi R-134 to its tagets. Mi R-134 inhibitor recombinant lentivirus containing GFP(or RFP) was constructed. Seventy-two hours after transfection to 293 T cells or neurons, about 80% of the cells with GFP(or RFP) expression were observed under fluorescence microscope. Quantitative real-time PCR detection showed that the mi R-134 expression in virus group was significantly lower than control group, which indicated that mi R-134 inhibitor recombinant lentivirus was packed successfully.2. Rat mi R-134 inhibited lentiviral vectors were stereotactically injected into bilateral hippocampus with EEG electrode. Two weeks later lithium chloride and pilocarpine were injected intraperitoneally in SD rats to induce status epilepticus(SE) and EEG monitoring of the rats were performed. Compared to the control virus group, the virus group(mi R-134 down regulation) had longer racine score Ⅴ lantency(P < 0.01), shorter duration of SE(P < 0.05), lower total EEG power(P < 0.001) with no significant difference in seizure severity. At 3 days after SE immunofluorescence staining in hippocampus showed that the virus group(inhibiting mi R-134) had more Neu N-positive cells and less Tunel-positive cells than the control virus group.3. The expression of mi R-134 was significantly up-regulated at 72 h after low-Mg2+ induced SE-like electrographic activities(P < 0.05). Down-regulation of mi R-134 expression by lentivirus decreased SE-like electrographic activities(P < 0.001) with more live cells(P < 0.001).4. Compared with the control virus group, the neuronal dendritic spines density in the mi R-134 inhibitor recombinant lentivirus group was decreased in the SE model that induced by low-Mg2+ extracellular fluid(P < 0.05). Conclusion1. In lithium-pilocarpme induced rat SE model, down-regulation of mi R-134 expression by lentivirus can relieve SE and exert neuroprotective effect.2. In the SE model that induced by low-Mg2+ extracellular fluid, down-regulation of mi R-134 expression by lentivirus can decrease SE-like discharge frequency and exert neuroprotective effect.3. Mi R-134 may play an important role in remodeling of dendritic spines.