The Progressive Depolymerization With A Transient Aggregation Of Filamentous Actin Cytoskeleton In The Hippocampal Neurons After Pilocarpine-induced Status Epilepticus

Objective: Temporal lobe epilepsy (TLE) is characterized by delayed, butpersisting loss of hippocampal neurons as well as extensive and persistent gliosis inthe limbic system. A positive correlation between the extent of cell death and the onsetof spontaneous seizures has been suggested in some animal TLE models, however thecellular processes mediating the delayed neuron death are still incompletelyunderstood. Since the maintenance of the shapes and functions of neurons is highlydependant on filamentous actin (F-actin) cytoskeleton, the present study is aimed toclarify the tempal and spatal changes of F-actin in the hippocampus afterpilocarpine-induced status epilepticus (SE).Method：A pilocarpine mouse model was used, and the methods includedhistological and TUNEL evaluation of cell loss, immunocytochemical analysis of glialreactions, and phalloidin detection of F-actin.Results:（1）Atotal of157ICR male mice are involved in this experiment, including135in the experimental group, and22in the control groups. About90mice developed theSE, among them,74mice survived with the survival rate of54.8%, and the death rateis10.4%. Five survived mice for2months were found to display epileptic seizuresand hippocampal sclerosis.（2）Nissl staining: In pilocarpine group6h PSE, the number of cells in the hiluswas significantly reduced. At PSE1d, the neuronal cells in CA1-CA3experiencedsignificant reductions (P<0.001) while at2-14d PSE, neuronal cells continued toreduce (P<0.001). At1d-14d PSE, there were almost no cells left in the hilus, but thedentate cells were less affected.（3）TUNEL staining showed that at1d PSE, the pyramidal cells in the hiluswere positively stained while at2-3d PSE we observed many apoptotic cells in the CA1-CA3areas. However, at5d-14d PSE, the number of cells was reducedsignificantly.（4）As compared with the control group, the fluorescence intensity for F-actin inCA1-CA3areas weas decreased, so did the F-actin in the dentate hilus. At7-14d PSE,there were almost no positive granules in these regions. According to the statisticalanalysis, the positive areas per square meter are significantly reduced.（5）As compared with the control group, the staining of astrocytes was increasedat1d PSE and reached the peak at7d PSE. It remained relatively high level on the14thday. According to the statistical analysis, the positive area per square meter areobviously increased (P<0.01).（6）As compared with the control group, the staining of astrocytes was increasedat1d PSE and reached the peak at7d PSE. It remained relatively high level on the14thday. According to the statistical analysis, the positive area per square meter areobviously increased (P<0.01).（7）After the double staining of F-actin and Calcium-binding protein D28K, wesaw that the positive granules of F-actin were close to the mossy fibers. The doublestaining of F-actin and synapsin I showed that the positive granules of F-actin wereclose to the end of the mossy fiber. The immunofluorescent staining of the PSD95showed that PSD95and F-actin were highly overlapped.（8）In vitro experiments, the group dealt with pilocarpine (5mM) showed a greatreduction in the dendrites and spines.The labeling of F-actin was significantlydecreased. In the dead neurons, F-actin was found to be completely lost.Conclusions:The present study showed that F-actin continued to gradually depolymerizeafter pilocarpine treatment, consistent in time course with the hippocampal neuronaldeath. The spatial changes of F-actin were found to exhibit common features amongdifferent hippocampal subfields. Firstly, F-actin puncta became decreased in number.Accompanying the nearly complete disappearance from dendritic spines, F-actin wastransiently aggregated in dendritic shafts and neuronal cell bodies, and was finally completely lost in the degenerated neurons.The progressive reduction with a transient aggregation of F-actin was alsoconfirmed in cultured hippocampal neurons, and may be a common molecularmechanism shared by all the eukaryotic cells during apoptosis. These observationsprovide some insights into a cytoskeletal mechanism that may link the delayed cellloss during epileptogenesis.