| Objective To observe the effects of electrical stimulation on hippocampus of α5Subunit of extrasynaptic GABAAreceptor in the kainic acid induced epilepsy rats, and toexplore the better therapeutic parameters and possible mechanism of electrical stimulation onhippocampus to treat the epilepsy.Methods1.Animal grouping: healthy male SD rats, were randomly divided into5groups: epilepsy group, sham operation group, LFS group, HFS group, normal control group.2. Established kainic acid in rats kindling model: the bipolar stimulating electrodesimplanted into the left hippocampus CA1region assisted by stereotactic, the injection cannulawas implanted into the right hippocampus CA3region. Postoperative7d with a micro-syringe injection of kainic acid0.6ul; After30d, epileptic seizures of rats into chronicepileptic period, beginning8hours a day (8:00-16:00) continuous observation of rat seizures,EEG of rats with monitoring.3. Seting the electrical stimulation parameters:after modeling of45d give electricalstimulation treatment; stimulation parameters: LFS treatment group (two-way square wavepulse,30min, pulse width1ms, strength100μ A, frequency1Hz, stimulus1times A day for2weeks), HFS treatment group (two-way square wave pulse,30min, pulse width0.2ms,amplitude5V, frequency130Hz, stimulus1times A day for2weeks), sham operation groupwithout any stimulation.4. Rats were killed after ether anesthesia, the brain and hippocampus tissue specimens,the application of the Nissl staining, q-PCR, Western blot detection between groups of α5 subunits relevant mRNA and protein expression in hippocampus. Data were analyzed byone-way ANOVA, between the two two groups were compared with LSD-t test, P <0.05hadsignificant difference.Results1.kainic acid kindling rats were successfully reached class Ⅴ attack, moldingsuccess rate was67%. After acute period of1~2d, the rat will no longer appear seizures, butincreased excitability, the rats into the stationary phase; After about30d rats appear repeatedspontaneous Ⅰ~Ⅲ seizures, occasionally Ⅳ~Ⅴ episodes, namely into the chronic phase.Before electrical stimulation treatment of rats model, seizures were not significant betweenrats epileptic seizure frequency (P>0.05).2.After45d given hippocampal electrical stimulation in the treatment of first1~5dperiod, rats with high excitability, aggressive, occasionally visible facial spasms, chewrhythmicity and wet dog dithering attacks in the treatment process; but with the increase ofthe stimulation time, rats gradually manifested as quiet, no epileptic seizures, during treatment,LFS treatme nt group, HFS treatment group compared with the sham operation group andepilepsy, the number of seizures decreased significantly (P<0.01), but the LFS treatmentgroup compared with HFS treatment group the number of seizures has no statisticalsignificance (P>0.05), the sham operation group compared with epilepsy group s the numberof seizures was not statistically significant (P>0.05). Normal control group did not appearseizures.3. The findings of Nissl staining showed that the number of neurons of LFS treatmentgroup and HFS treatment group were obviously higher than that in epileptic and shamoperation group（P﹤0.01）, in which, there was no significant difference between LFS groupand HFS treatment group (P>0.05), sham operation group and epilepsy group compared withno statistical significance (P>0.05). There were significant differences in LFS treatment groupcompared with the normal control group (P <0.05), there were significant differences in HFStreatment group compared with the normal control group (P <0.01). 4.q-PCR showed that epilepsy group, sham operation group, LFS treatment group, HFStreatment group and normal control group, the expression of the α5subunit mRNA were0.30±0.16,0.31±0.16,0.74±0.20,0.82±0.16,1.10±0.23; Westen blot shows: the epilepsygroup, sham operation group, LFS treatment group, HFS treatment group and normal controlgroup, the α5subunit protein expression were0.22±0.08,0.26±0.08,0.75±0.09,0.74±0.09,0.88±0.09. Among the two groups comparison showed that LFS treatment group, HFStreatment group and sham operation group, epilepsy group between α5subunit mRNA andprotein comparative differences are significant (P﹤0.01), the LFS treatment group comparedwith HFS treatment group has no statistical significance (P>0.05), the sham operation groupcompared with HFS treatment group has no statistical significance (P>0.05), LFS treatmentgroup, the HFS group compared with normal control group were significant difference (P﹤0.01).Conclusions Hippocampal electrical stimulation can effectively inhibit the seizures,Low frequency stimulation and high frequency stimulation is also effective; Extrasynaptic α5subunit inhibition plays an important role in seizure in hippocampal stimulation. |
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