| Microfilaments and microtubules are the basis of neuronal structure.Neuron death is characterized by progressive degradation of both.Therefore,the use of microfilament and microtubule stabilizing drugs to stabilize the cytoskeleton of neurons may play a role in preventing and treating delayed neuronal death.After epilepsy and transient cerebral ischemia,both F-actin and microtubules were depolymerized,leading to delayed neuronal death.Our previous studies have shown that after status epilepticus,F-actin in dendritic spines decreased progressively,but F-actin in dendritic trunks and cells bodies is aggregated.Since F-actin attaches to microtubules in the dendritic trunk at the base of dendritic spine,we speculate that F-actin aggregation in epileptic brain may be due to the loss of anchorage and support of F-actin after dendritic trunk microtubule depolymerization.F-actin damage of dendritic spine directly causes neuronal excitability abnormalities,while microtubule depolymerization aggravates the damage of F-actin of dendritic spine.The pathological process of cerebral ischemia is similar to that of status epilepticus,but the successful rate of delayed death of hippocampal neurons by using cerebral ischemia model is higher and the repeatability is better.Therefore,this project intends to use both pilocarpine-induced status epilepticus and transient global cerebral ischemia model to further explore the dynamic changes of F-actin and microtubules after neuronal delayed death.We then used cytoskeleton stabilizing drugs to explore the effect of cytoskeleton drugs on delayed onset.This study is aimed to elucidate the cytoskeleton-mediated mechanism in the development of epilepsy and cerebral ischemia,and to explore the inhibitory effect of on delayed death by interfering F-actin or microtubules.The experimental methods and results are as follows.1.An experimental study of stabilizing F-actin cytoskeleton to reduce delayed neuronal deathIn this chapter,the pilocarpine-induced SE model was used to observe the changes of seizure time and seizure frequency in mice through behavioral experiments;neuron death and mossy fiber germination were observed by immunohistochemical experiments;glial cell proliferation and F-actin were observed by immunofluorescence staining experiments;and the changes of F-actin were observed by Western experiments.The expression of F-actin,cofilin and p-cofilin was further detected by blot method.? The spontaneous onset time and frequency of epilepsy in mice were observed by behavioral experiments.The results showed that after treatment with FK506,the spontaneous onset frequency and duration of single epilepsy in mice were significantly reduced.? Immunofluorescence results showed that FK506 inhibited the death of some hippocampal CA1 and CA3 neurons and reduced glial proliferation.The positive area of F-actin in treatment group and model group increased,and the depolymerization of F-actin slowed down slightly.? Immunohistochemical results showed that the germination of mossy fibers decreased after FK506 injection.Western blot results showed that F/G ratio and cofilin phosphorylation increased significantly after injection of FK506,indicating that FK506 reduced the degradation of F-actin to a certain extent.These results suggest that calcineurin inhibitor FK506 can only alleviate epileptic symptoms to a certain extent,but the protective effect on the degradation of F-actin and delayed neuronal death is not obvious,so we speculate that there are other reasons related to delayed neuronal death,so the effect of stabilizing F-actin alone is not clear.Obviously,it provides a theoretical basis for subsequent experiments.2.Changes of F-actin and microtubules during neuronal death after transient global cerebral ischemiaIn this study,both in vivo and in vitro ischemia model were used.Neu N staining and Fluoro-Jade B staining were used to detect delayed neuronal death.Transmission electron microscopy was used to observe neuronal death in hippocampal CA1 region.Laser confocal microscopy was used to observe the changes of F-actin and microtubule-related proteins.The expression of F/G ratio,cofilin,p-cofilin,beta-tubulin III,MAP2 and acetyl-a-tubulin protein in hippocampal CA1 and CA3+DG regions were detected by Western blot.The interaction between F-actin and cofilin,F-actin and p-cofilin,F-actin and drebrin A was detected by immunoprecipitation.? After the delayed neuronal death model was induced by 4-vo global cerebral ischemia.,the pyramidal cells in CA1 area of hippocampus began to die on the second day of ischemia,and became serious with the increase of time.The death of neurons reached the maximum on the seventh day of ischemia-reperfusion.The positive area of F-actin in hippocampal CA1 began to decrease on the second day after ischemia,and reached the lowest on the seventh day.After ischemia,F-actin lost its binding to Drebrin a in dendritic spines,resulting in the decrease of F-actin stability.On the other hand,cofilin activity increased significantly,which may be the direct cause of F-actin depolymerization.? Similar to F-actin depolymerization,neurotubulin ?-tubulin III,MAP2 and act ubulin also decreased after ischemia.However,CO location analysis showed that the microtubule components in astrocytes increased.Western blot showed that the protein levels of MAP2,?-tubulin III and acetly-?-tubulin decreased significantly after transient ischemia.? Under the condition of primary culture of hippocampal neurons,the apoptotic rate of neurons increased significantly after OGD/R for 1h and 1.5h,and the progressive degradation of f-acin and transient rod aggregation occurred before DNA breakage.Meanwhile,the expression of Drebrin a,cofilin and p-cofilin decreased significantly.On the other hand,the microtubules of neurons also decreased progressively,and the stability of microtubules decreased significantly.These results suggest that the F-actin in hippocampal neurons degrades after transient global cerebral ischemia,but there is transient aggregation in the dendritic stem.On the other hand,the microtubules of neurons also decreased progressively,and the stability of microtubules decreased significantly.3 Experimental study on the effect of stabilizing microtubule cytoskeleton on delayed neuronal deathIn this study,transient global cerebral ischemia model was used.Immune fluorescence,Fluoro-Jade B and Nissl staining were used to detect that neuron death after treatment with Epo D.(1)Fluoro-Jade B and Nissl results showed that before and after low dose Epo D(0.5mg/kg)injection,neuronal death in hippocampal CA1 region could be slightly inhibited,while high dose 3mg/kg Epo D treatment could not inhibit neuronal death.(2)Immunofluorescence staining showed that the decrease of microtubules and related proteins was slightly inhibited before and after low dose 0.5mg/kg Epo D,but not highe dose 3mg/kg Epo D.In this study,the results before and after administration were similar in rats,suggesting that low doses of Epo D only slightly reduced the number of hippocampal neuron death induced by ischemia,while high doses of Epo D resulted in additional neuron death outside the hippocampal CA1 region.Because the microtubule assembly characteristics of hippocampal CA1 neurons and dentate gyrus granulosa cells are quite different,microtubule interference drugs that destroy the microtubule assembly balance may have complex effects on different types of neuron populations.ConclusionIn conclusion,after epilepsy and transient cerebral ischemia,F-actin in dendritic spines of hippocampal neurons degraded progressively,while F-actin in dendritic trunks accumulated temporarily.On the other hand,the microtubules in neurons also degraded progressively,but the microtubule composition in astrocytes increased significantly.Corresponding results were also confirmed in vitro neuronal ischemia experiments.The structure of neurons is highly dependent on F-actin and microtubules.Both cytoskeletons are depolymerized after delayed neuronal death,suggesting that cytoskeleton-stabilizing drugs may have the effect of inhibiting neuronal death.To this end,FK506 and Epo D were used to explore the significance of strengthening F-actin and microtubules in the treatment of neuronal death.FK506 slowed down the process of F-actin depolymerization and neuronal death to some extent,and improved animal behavior to some extent.However,different doses of Epo D had opposite effects on neuronal death.The assembled characteristics of cytoskeleton in neurons are different in different brain regions.In addition to the differences of neuron subtypes,the assembly characteristics of microtubules vary greatly among different regions of specific neurons.Therefore,microtubule interfering drugs that interfere with the balance of microtubule assembly may have complex effects on different types of neuron populations.Through the above experiments,we have elucidated the changes of F-actin and microtubules after delayed neuronal death,and preliminarily explored the effect of cytoskeleton drugs on delayed neuronal death,which provides a new theoretical basis for revealing the cytoskeleton mechanism of delayed neuronal death. |
PDF Full Text Request