Cdc42Participates In The Pathogenesis Of Epilepsy Through GABA Receptor Channels

Part one: Expression of Cdc42in epileptic ratObjective: To investigate the expression pattern in hippocampus oflithium-pilocarpine kindled rat models.Methods:1. Forty-eight healthy adult male Sprague-Dawley rats were randomlydivided into one normal control group (n=8) and five experimental group(subgroups:1day,3days,7days,30days and60days after seizure, n=8ineach subgroup). Rats were treated with lithium chloride plus pilocarpine toinduce seizure and to develop temporal lobe epileptic rats.2. To detect Cdc42expression position and expression changes inhippocampus of lithium-pilocarpine kindled rat models by double-labeledimmunofluorescence, Immunohistochemistry and western bolt.Results:1. Expression position of Cdc42in kindled rats detected bydouble-labeled immunofluorescence: Cdc42was expressed in thecytoplasm and cell membrane of neuron but not in the astrocytes. 2. Expression pattern of Cdc42detected by Immunohistochemistryz:Cdc42light expressed in the cytoplasm and cell membrane of neuron incontrol group. In model group, Cdc42up-regulation appeared at1days,3days,30days,60days after kindled (P<0.05). And Cdc42was expressedin the cell membrane and cytoplasm of neurons in CA1, CA3and dentategyrus of the hippocampus from kindled rats.3. Expression pattern of Cdc42detected by western bolt: Cdc42up-regulation appeared at1days after kindled, and maintained at arelatively high level until60days, the highest level of Cdc42expressionoccurred at days3after kindled (P<0.05).Conclusions: Cdc42expression was significantly increased inlithium-pilocarpine kindled rat models. These changes might have a keyrole in the development of epilepsy.Part two The effect of Cdc42selective inhibitor ML141on thebehavior of the epileptic ratsObjective: To observe the effect of Cdc42on the behavior oflithium-pilocarpine kindled rat models by intrahippocampal injectionML141, the potent and selective inhibitor of Cdc42.Methods: Fifty-four ealthy adult male Sprague-Dawley rats wererandomly divided into one normal control group (n=9), one vehicle group (n=9) and four ML141group (subgroups: ML1413umol, ML14110umol,ML14120umol and ML14150umol, n=9in each subgroup). All thesegroups were pretreated with corresponding drug by intrahippocampalinjection before intraperitoneal inject pilocarpine to induce seizure.Results:1. Seizure latency: Following intraperitoneal inject pilocarpine, thestatus epilepticus (SE) was induced, then we began to observe the latencyof seizure. The differences in latency between20μM ML141,50μMML141and control and vehicle were statistically significant (42.34±13.09min in20μM ML141;66.68±8.37min in50μM ML141;22.22±8.57min in Normal;23.53±8.17min in vehicle., p<0.05).2. Racine score: We next investigated that ML141decreased theintensity of seizure by racine score during the different time post-injectionof pilocarpine. We measured the racine score at10min,20min and30min after the onset seizure. There was significant difference in racine scorebetween normal control group(3.00±0.71,4.00±0.87,4.89±0.33) andML14150umol group (0.56±0.53,1.00±0.82,1.78±0.97)(P<0.05) andbetween vehicle group (3.33±0.71,4.22±0.83,4.89±0.33) and ML14150umol group (P<0.05). And there was no significant difference betweennormal group and vehicle group (P>0.05).Conclusions: The effective dose of Cdc42selective inhibitor ML141lengthened the latency of seizure and decreased the intensity of seizure. Part there The mechanisms of Cdc42on epileptic seizureactivityObjective: To investigate the effects of Cdc42on lithium-pilocarpineinduced epileptiform discharges of rat models.Methods: Whole cell patch-clamp recording techniques were used toobserve the changes of Action potentials (APs), miniature EPSCs,miniature IPSCs and evoked-IPSCs in normal group, model group andML141group.Results:1. Action potentials (APs): There was significant differences infrequency of APs between normal group and model group (P<0.05). Afterbathing ML141(3μM) for about15min in circulatory ACSF, recordingsshowed significant decrease in frequency of APs comparing with modelgroup (P<0.05), but no significant changes in frequency of APs comparingwith normal group (P>0.05).2. Miniature EPSCs:(1) Amplitude: contrast to model group, theamplitude of mEPSCs were significant decrease in normal group (K-S test,P<0.05) and no significant changes in ML141group (K-S test, P>0.05).(2)Frequency: contrast to model group, the frequency of mEPSCs weresignificant decrease in normal group (K-S test, P<0.05) and no significantchanges in ML141group (K-S test, P>0.05). 3. Miniature IPSCs:(1) Frequency: contrast to model group, thefrequency of mIPSCs were significant increase in normal group andML141group (K-S test, P>0.05).(2) Amplitude: contrast to model group,the amplitude of mIPSCs were significant increase in normal group andML141group (K-S test, P>0.05).4. Evoked-IPSCs: There were significant decrease in amplitude ofeIPSCs between normal group and model group (P<0.05). Contrast tomodel group, there were significant increase of amplitude of eIPSCs inML141group (P<0.05).Conclusions:1. Effective dose of ML141decreased the frequency of actionpotentials in the CA1pyramidal neurons of lithium-pilocarpine kindled ratmodels hippocampal slices.2. Effective dose of ML141had no effect on the frequency andamplitude of miniature EPSCs in the CA1pyramidal neurons oflithium-pilocarpine kindled rat models hippocampal slices.3. Effective dose of ML141increased the frequency and amplitude ofminiature IPSCs in the CA1pyramidal neurons of lithium-pilocarpinekindled rat models hippocampal slices.4. Effective dose of ML141increased the amplitude of evoked-IPSCsin the CA1pyramidal neurons of lithium-pilocarpine kindled rat models hippocampal slices.