| Part one: The expression of Dock3in temporal lobe epilepsy patientsand rat epilepsy modelsObjective:Abnormal synaptic transmission and neural network affect thechanges of neuron excitability in the brain。 Dock3(Dedicator ofcytokinesis3) is a newly confirmed guanine nucleotide exchange factor(GEF) which belongs to the Docks family. Dock3is specifically expressedin neurons and accumulates in the growth cone. It was associated withneuron excitability and neurite growth via Rac1(Ras-related C3botulinumtoxin substrate1) activation. Dock3was found involved in abnormal neuralnetwork and synaptic transmission in the brain tissues. Here weinvestigated the expression profile of Dock3in patients with temporal lobeepilepsy (TLE) and experimental rat models. And to detect relationshipbetween Dock3and temporal lobe epilepsy.Methods:1. Adult male healthy Sprague–Dawley (SD) rats were used in thisstudy. All animals were randomly divided into control group(n=5) and the epilepsy groups (n=35). Then the epilepsy groups were divided into7sub-groups by the time after pilocarpine induced status epilepticus:6hours,24hours,72hours,1week,2weeks,1month and2months (chronicepilepsy state with spontaneous recurrent seizure).2. Brain tissue from24patients with TLE were randomly selectedfrom brain tissue bank. in contrast,12patients with no history of epilepsywho undergoing surgery because of head trauma were also obtained fromthe brain bank used as controls.3. Expression profile of Dock3were assessed byimmunohistochemistry, qRT-PCR (Quantitative real-time polymerase chainreaction) and Western blot analysis.Results:1. Immunofluorescence staining showed that Dock3was primarilyobserved in the plasma membrane and the cytoplasm in CA3, CA1area ofrat hippocampus and also found in the cortex of rat and human brain tissue.Statistic differences were found that the mean optical density (OD) value ofDock3was higher in the TLE group compared with the control subjects(p<0.05).2. Western blot analysis showed that the expression of Dock3washigher in the subjects with intractable TLE and epilepsy models comparedwith control group.3. The neuronal marker MAP2but not the astrocyte marker GFAP were co-expressed with Dock3.Conclusions:The expression of Dock3was significantly increased in patients withTLE and a pilocarpine epilepsy model. These results suggest that Dock3may participate in the development of epilepsy.Part two: The effect of dock3knockdown on epileptic seizure andepileptogenesisObjective:To further explorer the effect of Dock3in epilepsy and whether Dock3intervention could affect epilepsy behavior change, Dock3short hairpinRNA(shRNA) were administered to observe animal behavior changes inpilocarpine epilepsy model and PTZ kindling model.Methods:1. For intrahippocampal injection, C57/BL6adult male mice wereused, all animals were divided into3groups at random: control group withhippocampal injection of saline (Con); vehicle shRNA group(hippocampalinjection of vehicle virus) and Dock3shRNA group (hippocampal injectionof Dock3shRNA).2. Western blot analysis were used to detect the expression of Dock3after Dock3shRNA intrahippocampal injection to confirm that Dock3shRNA was effective. And immunofluorescence was also used toconfirmed that the neurons were infected in the hippocampus. 3. Animals were intraperitoneally injected with subconvulsive doses ofPTZ (35mg/kg). After each PTZ injection,the convulsive behavior wasobserved for1hour and scored according to Racine. The animals wereconsidered to be kindled after having had four consecutive classes4or5seizure. For pilocarpine epilepsy model, We observed the behavioralchanges within50-min after pilocarpine intraperitoneally. Racine scoreswere recorded every5min interval after pilocarpine injection. The latenttime (from pilocarpine injection to the time first score4or5seizure attackoccurred). During the chronic stage of epilepsy, The video system wereused to record seizure attack (Racine score4or5). for every animals, weaccumulate the seizure times for1week.Results:1. The expression of Dock3were found statistical decreased inanimals intrahippocampal injection Dock3shRNA at days5and6weekscompared with vehicle shRNA group and control group (p <0.05). Therewas no significantly difference between vehicle shRNA group and controlgroup (p>0.05). The immunfluresene analysis showed that EGFP wasmajor localized in the corpus callosum and hippocampus, especially inCA3region and dentate gyrus.2. The seizure class of the Dock3shRNA-treated group wassignificantly different compared to the control and vehicle shRNA-treatedgroups at the time points of4,7,8,10,11and14days(p <0.05). and the latent period in Dock3shRNA-treated group also prolonged significantlycompared to the control and vehicle shRNA-treated groups.3. In the pilocarpine epilepsy model, Dock3shRNA-treated miceexhibited significantly attenuated seizure classes after pilocarpineinjection. We also found that the frequency of spontaneous seizures wassignificantly decreased in the Dock3shRNA-treated mice compared tothe control and vehicle shRNA-treated groups during the chronic stageof the pilocarpine-induced epilepsy model (p <0.05).4. Timm staining scores of Dock3shRNA-treated mice showedsignificantly decreased compared with control and vehicleshRNA-treated groups.Conclusions:1. The expression of Dock3in the hippocampus was successfullysuppressed in vivo by Dock3shRNA at day5and6weeks.2. Knockdown of Dock3with Dock3shRNA in epileptic animalscould reduce seizure severity in the acute stage and decreased the SRStimes in the chronic stage in pilocarpine epilepsy model. Dock3alsoprolonged the process of kindling in PTZ kindling model.3. Inhibition of Dock3attenuate the severity of mossy fiber sprouting.Part three: The cell mechanisms of Dock3in epilepsyObjective:To explore the possible mechanisms of of Dock3involved in epilepsy. whole cell patch clamp were used to detect the effect of Dock3onelectrophysiological changes.Methods:1. Three groups with intrahippocampal injection of saline (Con),vehicle shRNA, and Dock3shRNA were included in the study. ACSFwithout magnesium were used in the study to detect seizure activitychanges on mice brain slices.2. Whole-cell patch clamp were performed to recorde the AP(actionpotential), mEPSCs(miniature excitatory postsynaptic currents) andeEPSCs(evoked miniature excitatory postsynaptic currents). The frequencyof AP, amplitude ratio of NMDAR/AMPAR, the amplitude and frequencyof mEPSC and eEPSC.Results:1. Compared with vehicle shRNA group or control group, thefrequency of AP significantly decreased (p<0.05), while no differenceswere seen between the vehicle shRNA group and control group(p>0.05).2. The effect of Dock3on mEPSC:(1) the effect on frequency: thefrequency in hippocample neurons of Dock3shRNA treated group wassignificantly decreased compared with vehicle shRNA group or controlgroup(p<0.05), statistical analysis showed no significant differencesbetween the vehicle shRNA group and control groups(p>0.05).(2) therewere no significantly difference of mEPSC amplitude in hippocample neurons in Dock3shRNA treated group compared with vehicle shRNAgroup or control group (p>0.05).3. The amplitude ratio of NMDAR/AMPAR in Dock3shRNA treatedgroup was significantly decreased (p<0.05), further analysis showed thatthe amplitude of NMDA mediated EPSCs in Dock3shRNA treated groupwas significantly decreased compared with vehicle shRNA or control group(p<0.05). while the amplitude of AMPA mediated EPSCs in Dock3shRNAtreated group showed no significant chang compared with vehicle shRNAor control group(p>0.05).Conclusions:1. Dock3shRNA inhibited the frequency of AP and attenuatesuppressed the hyperexcitability in hippocample neurons.2. Knockdown of Dock3with Dock3shRNA significantly decreasedthe frequency of mEPSCs.3. Knockdown of Dock3with Dock3shRNA effectively decreased theamplitude of NMDA mediated EPSCs but not AMPA mediated EPSCs.Part four: Rac1is required to the potential mechanisms of dock3involved in epilepsyObjective:To further explore the possible mechanisms of Dock3involved inepilepsy. We then detected the expression of Rac1, because Rac1is aspecific substrate of Dock3. To determine whether the effect of Dock3on epilepsy requires the activation of Rac1, Rac1inhibitor NSC23766wasused in rhe study. NSC23766intracerebroventricular injection was usedat the same time with Dock3shRNA, then timm staining and behaviorchanges were observed to explore the effect of Dock3in the absence ofRac1.Methods:1. A guide cannula was implanted into the right lateral ventricle, Rac1inhibitor NSC23766was intracerebroventricular microinjection (i.c.v) tothe brain every day for2weeks. Adult male SD rats were divided into3group: control group (intracerebroventricular injection of normal saline);NSC2376650μM group (intracerebroventricular injection of NSC2376650μM) and100μM group (intracerebroventricular injection of NSC23766100μM). For recombinant administration of Dock3shRNA and NSC23766,C57/BL6adult male mice were used, all animals were divided into3groups: NSC23766group (hippocampal injection of saline plus i.c.v.NSC23766); vehicle shRNA+NSC23766group(hippocampal injection ofvehicle virus plus i.c.v. NSC23766) and Dock3shRNA+NSC23766group(hippocampal injection of Dock3shRNA plus i.c.v. NSC23766).2. Animals were intraperitoneally injected with subconvulsive doses ofPTZ (35mg/kg) in saline everyday. After each PTZ injection,theconvulsive behavior was observed for1hour and scored according toRacine. The animals were considered to be kindled after having had four consecutive classes4or5seizure. For pilocarpine epilepsy model, Weobserved the behavioral changes within50-min after pilocarpineintraperitoneally. Racine scores were detected every5min. The latentperiod (from pilocarpine injection to the time first score4or5class seizureoccurred). During the chronic stage of epilepsy, The video system wereused to record seizure attack (Racine score4or5). for every animals, weaccumulate the seizure times for1week.3. After intrahippocampal injection Dock3shRNA with NSC23766,behavior changes were observed in the acute and chronic stage in thepilocarpine model. Western blot analysis and timm staining were used foranalysis of the Rac1-GTP expression and mossy fiber sproutingrespectively.Results:1. Western blot analysis showed compared with the control group,both Rac1and Rac1-GTP expression increased from the acute period untilthe chronic period (2m time point), and their expression profile presentedalmost the same. Statistical analysis showed statistical differences of theRac1/Rac1-GTP expression level between the experiment and controlgroups (p<0.05). Western blot analysis also showed that the expression ofRac1-GTP significantly decreased after NSC23766administration(p<0.05).2. in PTZ kindling model, NSC23766intervention did have a significant effect on the kindling time of epilepsy, the seizure class of theNSC23766-treated group exhibited significant differences compared withthe control and NS treated groups at the time points5,6,8-10,13-15,17days(p <0.05). in the pilocarpine epilepsy model, NSC23766significantlydecreased the seizure class in the acute stage, and also prolonged the latentperiod significantly.3. Western blot analysis showed that Dock3shRNA administrationsignificant decreased the expression of Rac1-GTP. However, no statisticaldifference was found between the vehicle shRNA and control group(p>0.05).3. when Rac1inhibitor NSC23766was used with Dock3shRNA at thesame time. The seizure classes in the acute stage and the frequency ofspontaneous seizures observed in the chronic stage in Dock3shRNA-treated mice showed no significantly differences compared withthe control and vehicle shRNA-treated groups after pilocarpineinjection(p>0.05). Timm staining analysis also indicate that no differenceswere found among these3groups(p>0.05).Conclusions:1. The expression of Rac1/Rac1-GTP were found significantlyincreased in patients with TLE and a pilocarpine epilepsy model. Dock3shRNA administration significant decreased the expression of Rac1-GTP.These results suggest that Dock3/Rac1may participate in the development of epilepsy.2. The activation of Rac1was decreased after dock3shRNAadministration. Meanwhile, the severity of behavior change and mossyfiber sprouting also decreased. When Rac1inhibitor NSC23766were used,no differences were seen both in behavior change and timm scores. Theresult indicate that the effect of Dock3on epilepsy requires the activationof Rac1. |
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