| PART ONE: The expression of mi RNA-137 in patients with temporal lobe epilepsy and mouse models of epilepsy.Objective: To investigate the expression profiles of mi RNA-137 in brain tissues of temporal lobe epilepsy patients and two different epilepsy mouse models(pilocarpine- and pentylenetetrazole-induced mouse models of epilepsy).Methods:Temporal neocortex tissues from 18 intractable temporal lobe epilepsy patients were chosen at random from our epilepsy brain tissue bank.Control samples were randomly collected from 12 craniocerebral trauma patients undergoing surgical removal temporal neocortex for treatment.Adult male C57BL/6 micewere used to establish epilepsy models by being injected pilocarpine or PTZ. In the pilocarpine-induced mouse model of epilepsy, the mice detected spontaneous recurrent seizures(SRS) were defined as the epilepsy group, and those without SRS were defined as the controls. In PTZ-kindled epileptic mouse model, fully kindled mice were defined as the epilepsy group, and those without fully kindling weredefined as the control group. Cortex and hippocampus tissues were obtained from the epilepsy and control groups. q RT-PCR was used to investigate the expression level changes of mi RNA-137 in brain tissues between epilepsy group and control group.Results:1. The mi RNA-137 expression level in temporal neocortex tissue of temporal lobe epilepsy(TLE) patients was significantly lower than that in control patients(P<0.05);2. In pilocarpine-induced epileptic mouse model, mi RNA-137 expression both in hippocampus and cortex downregulated in epilepsy group compared to control group(P<0.05);3. In PTZ-kindled epileptic mouse model, mi RNA-137 expression both in hippocampus and cortex of epilepsy group were suppressed in fully kindled epilepsy group while not in the controls(P<0.05).Conclusion:Expression of mi RNA-137 decreased both in TLE patients and two different epileptic mouse models. These results indicated that mi RNA-137 may have a close relationship with epileptogenesis.PART TWO: The effect of mi RNA-137 in vivo intervention on animal epileptic behaviors.Objective: To uncover the effect of mi RNA-137 in vivo intervention byintrahippocampal injection of mi RNA-137 special agonist(agomir)and inhibitor(antagomir)upon the animal epileptic behaviors.Methods:1. Adult male C57BL/6 mice were randomly divided into five groups:control group, agomir NC group, agomir group, antagomir NC group and antagomir group. Saline, agomir scrambled NC 0.2nmol, agomir 0.2nmol,antagomir scrambled NC 0.8nmol and antagomir 0.8nmol were injected to hippocampus, respectively.2. q RT-PCR and laser confocal fluorescence microscope were used to investigate the intervention efficiency of agomir and antagomir by stereotaxic intrahippocampal injection.3. Animals were injected intraperitoneally with pilocarpine(320mg/kg)to establish pilocarpine-induced epileptic mouse model. During the chronic phase of pilocarpine-induced epilepsy, we observed the latency to the first SRS and accumulated the SRS times for 7 days. Meanwhile, animals received a subthreshold-dose PTZ(35mg/kg.day) to establish PTZ-kindled epileptic mouse model by intraperitoneal injection. We observed the latency to fully-kindled and the seizure class scores according to Racine score for 1 hour after each injection.Results:1. The expression level of mi RNA-137 in hippocampus were higher in agomir group than that in control group at 3 days,1 week, 2 weeks and 4weeks respectively after intrahippocampal injection of mi RNA-137 agomir(P<0.05). On the other hand, the expression level of mi RNA-137 in hippocampus were lower in antagomir group than in control group at 3days,1 week, 2 weeks and 4 weeks respectively after intrahippocampal injection of mi RNA-137 antagomir(P<0.05). The laser confocal fluorescence microscope showed that FAM conjugated mi RNA-137 agomir or antagomir was localized in hippocampus of mice.2. In the pilocarpine-induced epileptic mouse model, we observed that the latency to SRS was delayed and the number of SRS was decreased in agomir group compared to control group and agomir NC group(P<0.05).On the other hand, compared with the antagomir NC and control groups,the latency to SRS was shorten and the number of SRS was larger in antagomir group(P<0.05);3. In the PTZ-kindled epileptic mouse model, we observed that the latency to fully-kindled was prolonged in agomir group than that in control and agomir NC groups(P<0.05). Compared with control group, the seizure class of the agomir group was lower at the time points of 5-12 days(P<0.05). Meanwhile, the latency to fully-kindled was shorten in antagomir group compared with control group and antagomir NC group(P<0.05).Compared with control group, the seizure class of the antagomir group was increased at the time points of 4-9 days(P<0.05).Conclusion:1、The expression of mi RNA-137 in hippocampus was significantly increased after mi RNA-137 agomir intrahippocampal injection;mi RNA-137 antagomir intrahippocampal injection specifically inhibited the expression of mi RNA-137 in hippocampus.2、The mi RNA-137 in vivo intervention could change the latency and severity of seizure in pilocarpine-induced and PTZ-kindled mouse models of epilepsy.PART THREE: The effect of mi RNA-137 in vivo intervention on excitability of pyramidal neurons in hippocampus.Objective: To explore the effect of different expression levels of mi RNA-137 by intrahippocampal injection of mi RNA-137 special agonist and inhibitor on the excitability of the pyramidal neurons in the hippocampus of mice.Methods:1. Male C57BL/6 mice were included in this study. 72 hours after injected with saline, or agomir scrambled NC, or antagomir scrambled NC,or agomir, or antagomir in hippocampus, the mice were prepared to brain slices.2. Mg2+ free a CSF perfusion were used to induce epileptiform discharges in mice brain slices. The whole-cell patch clamp technique was used to record the changes of electrophysiological pattern in pyramidalneurons of CA3 region. The frequency of action potentials(AP), the frequency and amplitude of miniature excitatory postsynaptic currents(m EPSCs) and miniature inhibitory postsynaptic currents(m IPSCs),the values of paired-pulse ratio(PPR) were recorded and analysed.Results:1. The frequency of AP was decreased in agomir group compared with the groups of control and agomir NC. While the frequency of AP was increased in antagomir group compared with groups of control and antagomir NC(P<0.05);2. The frequency of m IPSC was upregulated in agomir group compared with the groups of control and agomir NC. While the frequency of m IPSC was downregulated in antagomir group compared with groups of control and antagomir NC(P<0.05). However, there were no statistical differences in the amplitude of m IPSCs among the five subgroups(P>0.05);3. Compared with groups of control and agomir NC, the frequency of m EPSCs in agomir group was no statistical difference(P>0.05);Meanwhile, antagomir group had no statistical difference in frequency of m EPSCs compared to antagomir NC and control groups(P>0.05). Similar to frequency of m EPSCs, there were no statistical difference in amplitude of m IPSCs in agomir group compared with control group and agomir NC group(P>0.05); Antagomir group also had no statistical difference inamplitude of m EPSCs compared to antagomir NC and control groups(P>0.05);4. Compared with control group and agomir NC group, the value of PPR in agomir group significantly decreased in pyramidal neurons of hippocampus(P<0.05).Conclusion:1、mi RNA-137 in vivo intervention could change the excitability of the pyramidal neurons in the hippocampus of mice.2、mi RNA-137 in vivo intervention might regulate the frequency of m IPSCs by governing the release of presynaptic inhibitory neurotransmitters. |
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