| The long-lasting plastic and structrural change of synaptic network in epilepticbrain is thought to be one of pathologic machanisms of epileptogenesis. The structuralremodeling of synapses is driven by changes of actin dynamics. However, due to lackof effective detection means, there are very few investigations on the filamentous actin(F-actin) mechanism in epilepsy. We used phalloidin to label F-actin specifically,together with pre-/postsynaptic labeling and protein techniques, to study the changesof F-actin during the pathological process of post status epilepticus model and kindlingmodel. By clarifying the F-actin mechanism underlying the epileptic brains, our studywas aimed to provide a novel target for therapy of epilepsy.Objective By clarifying the F-actin mechnism underlying the epileptic brains, ourstudy was aimed to provide a novel target for therapy of epilepsy.Method We used the post status epilepticus model induced by pilocarpine and thechemical kindling model induced by Pentylenetetrazol (PTZ) in C57BL/6mice. In thisstudy, we used phalloidin to label F-actin specifically, the antibodies to synapsin I andPSD95to recognize the pre-/postsynaptic region, the double staining ofF-actin/synapsin I and F-actin/PSD95to analyze the distribution of F-actin inpre-/postsynaptic region, together with Western blot protein techniques of F/G ratio,PSD95、synapsin I、cofilin and p-cofilin, to clarify the F-actin mechnism underlyingthe epileptic brains.Results1. In the post status epilepticus (SE)models induced by pilocarpine:(1) Model evaluation: In the pilocarine group,62.5%（100/160） mice meet thestandard of SE and19of them survived. The rate of success was11.88%（19/160） andmortality was50.62%（81/160）。All the mice survived from SE had spontanous seizures after2months.(2) Altered F-actin intensity in pilocarpine model during acute period, latent periodand chronic period: the fluorescence intensity of F-actin was dramatically reduced inthe CA3stratum lucidum in acute period(6h post SE), while recovered in latent period(48h and10days post SE). In chronic period (2months post SE), F-actin was reducedagain in the CA3stratum lucidum, accompanied by abnormal morphology anddistribution.(3) Results of Nissl, Timm and GFAP staining in chronic period: the results ofNissl Staining showed cell loss was obviously found in the CA1-CA3regions, while asignificant increase of cells in DG region was found in the hippocampus of post SEmice (p<0.05). As compared to those in the control, positive granules of Timm stainingwas obviously seen in the supragranular region in post SE group (p<0.05). GFAPstaining was dramatically increased in post SE group (p<0.05).(4) Results of F-actin labeling and F/G ratio detection in chronic period: At lowpower magnification, the intensity of the stratum lucidum of CA3region was slightlydecreased in post SE models. At high power magnification, the F-actin puncta wereobviously increased in size but decreasd in number in post SE mice. As compared tothose in the control, the positive area and the number of puncta per square micrometerwere significantly smaller (p<0.05), but the size per punctum was significantly largerin the post SE mice. Quantitative analysis of Western blot results further showed thatthe F/G ratio was significantly down-regulated in the post SE mice (p<0.05).(5) Results of synapsin I staining and Western blot in chronic period: At lowerpower magnification, the intensity of the stratum lucidum of CA3region was slightlydecreased in post SE model. At high power magnification, synapsin I positive punctawere also obviously increased in size but decreasd in number in post SE mice. Thepositive area per square micrometer did not show much diference between the twogroups (p>0.05), but the size per punctum was significantly larger and the number ofpuncta per square micrometer was significantly smaller in the post SE mice. Westernblot data of synapsin I did not show much diference between the two groups either (p>0.05).(6) Results of PSD95staining and Western blot in chronic period: As comparedto those in the control, the positive area of PSD95per square micrometer wassignificantly decreased in the post SE mice (p<0.05) and Western blot data alsosuggested that PSD95was significantly down-regulated in the post SE mice mice(p<0.05).(7) Double labeling results of F-actin/synapsin I and F-actin/PSD95in chronicperiod: The double labeling showed the total co-localized area of F-actin and synapsinI was decreased but the co-localized area of per punctum was increased in the stratumlucidum of CA3region in post SE mice. Quantitative analysis showed significantdifference between the two groups.(p<0.05). The double labeling of F-actin/PSD95also showed the positive puncta for PSD95were highly overlapped with those forF-actin, but the distribution of PSD95was not identical to F-actin.2. In PTZ chemical kindling models:(1) Model evaluation: In the daily injected group,80%(12of15) of the micemeet the standard of kindling and it averagely took19.7+/-5.3days (n=12) to besuccessfully kindled. In the group injected every48hrs, only46.7%(7/15) of the micewere successfully kindled, and the average time was30.0+/-20.3injects (n=7).(2) Results of Nissl and Timm staining: The results of Nissl Staining showedlimited cell loss in the CA1-CA3regions in the hippocampus in PTZ-kindled miceand quantitative analysis of the CA3b region indicated that PTZ kindling induced onlya slight loss of cells in the C57BL/6mice (p>0.05). Positive granules of Timm stainingwere rarely seen in the supragranular region or in the CA3pyramidal cell layers in thetwo groups. A slight increase of staining intensity was observed in the stratum lucidumof CA3region in the kindled mice and quantitative analysis of the optical density inthis region demonstrated that the difference was statistically significant (p<0.05).(3) Results of F-actin labeling and F/G ratio detection: At low powermagnification, almost no obvious change was discerned between the two groups in thestratum lucidum of CA3region. At high power magnification, the F-actin puncta were obviously increased in number in the PTZ-kindled mice. As compared to those in thecontrol, both the positive area and the number of puncta per square micrometer weresignificantly larger in the kindled mice (p<0.05). Quantitative analysis of Western blotresults further showed that the F/G ratio was significantly up-regulated in the kindledmice (p<0.05).(4) Results of synapsin I staining and Western blot: At lower power magnification,the punctuate labeling pattern seemed not much different between the two groups, butexamination of higher power images showed that the positive area and the size ofindividual puncta were obviously increased in the kindled mice as compared to thosein the control (p<0.05). Consistently, Western blot data also suggested that theexpression of synapsin I was significantly up-regulated in the kindled mice (p<0.05).(5) Results of PSD95staining and Western blot: As compared to that in thecontrol, the positive area per square micrometer was significantly decreased in thePTZ-kindled mice (p<0.05) and Western blot data also suggested that PSD95wassignificantly down-regulated in the PTZ-kindled mice (p<0.05).(6) Double labeling results of F-actin/synapsin I and F-actin/PSD95: The doublelabeling of F-actin/synapsin I showed in the stratum lucidum of CA3region, phalloidinlabeling was located in close proximity to the puncta positive for synapsin I. A narrowband of the F-actin puncta was localized peripherally within the synapsin I-positivestructure and quantitative analysis by measuring the yellow area per square micrometershowed that the presynaptic F-actin was significantly increased in the stratum lucidumin the kindled mice, as compared to that in the control (p<0.05). The double labeling ofF-actin/PSD95showed the positive puncta for PSD95were highly overlapped withthose for F-actin, but the distribution of PSD95was not identical to F-actin.Conclusions1. F-actin and synapsin I were co-localized in the presynaptic region of CA3stratum lucidun in C57BL/6mice.2. In post SE models induced by pilocarpine in C57BL/6mice，the fluorescenceintensity of F-actin was dramatically reduced in the CA3stratum lucidum in acute period, while recovered in latent period. In chronic period, F-actin was reduced again inthe CA3stratum lucidum, accompanied by abnormal morphology and distribution.3. There were significant cell loss, mossy fiber sprouting and gliocyte proliferationin the chronic period of pilocarpine induced post SE model.4. The rearrangement of F-actin mainly occurred postsynaptically in the CA3stratum lucidum in the chronic period of pilocarpine induced post SE model and wasprobably related to the spontaneous recurrent seizures.5. There were no significant cell loss and mossy fiber sprouting in PTZ kindledC57BL/6mice.6. In PTZ chemical kindling models of C57BL/6mice, F-actin rearrangeddifferentially in pre/post synaptic region in CA3stratum lucidum. F-actin wasincreased presynaptically and decreased postsynaptically, which seemed to be involvedin seizures susceptibility. |
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