Empirical Study On The Related Mechanism Of μ-calpain And Neuronal Programmed Cell Death In Hippocampus Of Status Epilepticus-Rats

Epilepsy is one of the most common nervous system diseases,of which morbidity rate is 5‰～7‰.There are about 35 million patients in the world and 6.5 million patients in China that suffer from this disease.Status epilepticus(SE)is a serious status of epilepsy and is defined as two or more sequential seizures without full recovery of consciousness between seizures,or more than 30 minutes of continuous seizure activity.SE can electively induce hippocampal neuron death,and the death promoted the formation and development of epilepsy.So,people increasingly pay attention to neuroprotective therapy for epilepsy patients.The evidence sufficiently indicated that SE could induce obvious neuron damage,especially in limbic system of animal and human.Necrosis is generally thought to be the main form of cell death of cerebral cortex after epilepsy;in addition,apoptosis also plays an important role in brain damage induced by epilepsy.Calpain,calcium-activated non-lysosomal protease,isáCa²⁺-dependent cysteine proteinase,which extensively exists in nerve cell and has two kinds of tissue-nonspecific forms:μ-calpain(calpain-1)and m-calpain(calpain-2).μ-calpain is ubiquitously distributed in neurons,while m-calpain mainly in inhibitory interneuron and astrocyte.Both isoforms are modulated by calpastatin,an endogenous calpain inhibitor.High concentration free-Ca²⁺activates calpain and activated calpain can hydrolyze cytoskeletal protein,kinase and phosphatase,some signal transducer and transcription factor,and leads to cell death in the end.Calpain involved in neuronal death appears in many kinds of neurogenic diseases, such as cerebral ischemia,spinal injury,Alzheimer disease,and so on.The conventional view is that the activation of calpain is responsible for neuron necrosis, but recent evidence showed calpain also led to neuronal programmed cell death (PCD).The relation between chaplain's activation and PCD was firstly found in thymocyte,afterwards,this correlation was also found in cell PCD induced by many etiological factors.In focal cerebral ischemia and brain transient ischemia-reperfusion of rats,in CA1 area of hippocampus,calpain was extensively activated,morphous of neuron changed,DNA gragmentation and PCD appeared.Specific inhibitor of calpain could inhibit PCD,which demonstrated that chaplain's activation was responsible for the process of PCD.Whether calpain participates in neuronal PCD after SE and its mechanism,there is no coincident view.The observations suggest that KA evokes excitotoxicity in hippocampus by promoting Bid activation,nuclear translocation of apoptosis inducing factor(AIF)and endonuclease G,DNA fragmentation and nuclear condensation that were significantly augmented by calpastatin deficiency.These strongly suggest that calpain plays a pivotal role in the excitotoxic signal transduction cascade leading to DNA fragmentation.However,considering that the results in wild-type mice given intrahippocampal KA were similar to those of calpastatin-overexpressing mice.So,the precise roles of calpain in seizure-induced neuronal PCD in rat with "normal" levels of calpain and calpastatin need to be further investigated.The study was designed to establish lithium chloride-pilocarpine-induced status epilepticus(LPSE)in rats and examine pathological character of neuronal damage after SE,activity ofμ-calpain and some molecules related with PCD.The study interfered in SE model by using calpain inhibitor-MDL28170,and examined expressions of proteins and genes related with PCD and signal path mediated byμ-calpain,and evaluated protection for hippocampus neuron of MDL28170. Part 1 Hippocampual damage and neuronal programmed cell death in lithium chloride-pilocarpine-induced status epilepticus-ratsObjective:To investigate ethology,clectrophysiological and pathological changes of LPSE rats,explore hippocampal neuronal injury induced by seizures,and observe the existence of neuronal PCD after epilepsy.Methods:1.Male Wister rats were divided into control group and 6 h,12 h,1 d,3 d,5 d,7 d after seizures.The SE groups were treated with lithium chloride and pilocarpine.SE was terminated with intraperitoneal injection of diazepam until it lasted for 2h.2.The rats were given EEG examination before and after seizures.3.Rats were intracardially perfused with 4%paraformaldehyde and the brains were removed at different time-points after seizures.Hippocampal pathological changes were observed with haematoxylin & eosin(HE)and Nissl staining.4.To observe neuron PCD of hippocampus with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling(TUNEL).Results:1.The incidence rate of SE induced by lithium chloride and pilocarpine was 91.7%. EEG showed spike discharge at acute stage and normal at latent period.2.The surviving neurons showed round and palely stained nuclei.From 6h to 7d after SE,hippocampal neuron damage was found.This damage showed meanwhile, the dead neurons in hippocampus show pyknotic nuclei and shrunken plasma body, survival neurons decreased compared to control group(p＜0.05)3.TUNEL positive neuron slightly expressed in hippocampus on 6 h after LPSE rat and predominately increased on 3 d and continued till 7 d after SE.The number of TUNEL-positive neurons were more than the control group(P＜0.05).Furthermore,the TUNEL-positive neurons of 3 d,5 d and 7 d were more than these of 6 h,12 h and 1 d(P＜0.05).Conclusions: 1.Lithium chloride-pilocarpine successfully induced SE in rat model according to the changes of ethology and EEG after seizures.2.LPSE could result in hippocampal neuronal damage.3.LPSE could result in neuron PCD of hippocampus.Part 2 Empirical study ofμ-calpain involved in haippocampal neuronal programmed cell death in lithium chloride-pilocarpine-induced status epilepticus-ratsObjectiveTo investigate the participation ofμ-calpain in hippocampal neuronal PCD in LPSE-rat,and the neuroprotective role of MDL-28170,the inhibitor ofμ-calpain,in hippocampal neuronal injury.Methods1.Male Wistar rats were randomly divided into 7 groups:control group,and 6 h,12 h,1 d,3 d,5 d,7 d groups after seizure.The animals received lithium chloride and pilocarpine to induce SE that were terminated by an injection of diazepam until continued for 2 hours.We divided the 3 d group into four subgroups(pilocarpine, pilocarpine+saline,pilocarpine+MDL-28170 and control)to study the function of MDL-28170,the inhibitor ofμ-calpain.2.Rats were intracardially perfused with 4%paraformaldehyde in phosphate-buffered saline under anesthesia at different time-points after seizures.The expression of activatedμ-calpain and caspase-3 were detected by immunohistochemistry(IHC),and observe the impact of MDL-28170 on their expression.3.Rats were decapitated at different time-points,and hippocampal protein was obtained,the expression ofμ-calpain,caspase-3 andα-â…¡-spectrin(αSpâ…¡)were detected by Western blotting,and observe the impact of MDL-28170 on their expression.4.Nissl staining showed the protective effect of MDL-28170 against the hippocampal neuronal damage after SE.5.TUNEL showed the protective effect of MDL-28170 against the hippocampal neuronal PCD after SE.Results1.μ-calpain was activated at 6 h after SE,and reached the maximum at 3 d,but caspase-3 was primarily activated at 1 d and reaches the maximum at 5-7 d.2.Rats that received MDL-28170 showed more neuron survival compared with pilocarpine group and pilocarpine+saline group(P＜0.05).3.Rats that received MDL-28170 showed decreased level of PCD compared with pilocarpine group and pilocarpine+saline group(P＜0.05).4.MDL-28170 can attenuate the expression ofμ-calpain and calpain-specific fragment ofαSpâ…¡(145kD),but have no effect on caspase-3 and the caspase-3-specific fragment ofαSpâ…¡(120kD).Conclusions1.Bothμ-calpain and caspase-3 participate in the process of hippocampal neuronal PCD andμ-calpain functions in the early phase,while caspase-3 in the late phase.2.MDL-28170 protects hippocampal neuron after pilocarpine-induced seizure against PCD through the inhibited effect onμ-calpain.Part 3 The study ofμ-calpain and its signal pathway involved in hippocampal neuronal programmed cell death in lithium chloride-pilocarpine-induced-status epilepticus-rats ObjectiveTo investigate the signal transduction pathway that involved in the process of hippocampal neuronal PCD in LPSE-rats throughμ-calpain.Methods1.Male Wistar rats received lithium and pilocarpine to induce SE.Seizures continued for 2 hours and were terminated by an injection of diazepam.Rats were randomly divided into 7 groups:control and 6 h,12 h,1 d,3 d,5 d,7 d after SE to analyze the expression of related proteins and their dynamic changes by Western blotting.Three days-rats after SE were divided into pilocarpine,pilocarpine+saline, pilocarpine+MDL-28170,pilocarpine+ZVF and normal control groups.MDL-28170 is a selective inhibitor ofμ-calpain(before and after SE,i.p.)and ZVF,Z-VAD (OMe)-fmk,is a caspases pan-inhibitor(before and after SE,i.c.v.).2.The hippocampi were obtained and stored at -80℃.3.The protein expression of Bid and alteration of AIF,cytochrome c in different cellular components in LPSE and control rats by Western blotting.4.The mRNA expression ofμ-calpain,caspase-3,caspase-8,caspase-9,Bid and AIF was detected in hippocampi of 3 d after LPSE and control rats by reverse transcriptase-polymerase chain reaction(RT-PCR),in the meantime,the effects of MDL-28170 and ZVF on their expression were observed.5.The proteins expression of Bid,AIF and cytochrome c was detected in hippocmapi of 3 d after LPSE and control rats by Western blotting,and the effects of MDL-28170 and ZVF on their expression were observed.Results1.The protein expression of tBid(activated form of Bid),nuclear translocation of AIF and release of cytochrome c from(?)itochondria in hippocampi of 12 h,1 d,3 d,5 d and 7 d after LPSE-rats were significantly deviation compared with that of control rats(P＜0.05).The expression of tBid,nuclear translocation of AIF and release of cytochrome c reached maximum at 3 d after LPSE,and tapered but showed more obviously than that of control rats(P＜0.05).2.The mRNA expression ofμ-calpain,Bid and AIF in hippocampi of 3 d after LPSE-rats up-regulated(P＜0.05),however,that of caspase-3,caspase-8 and caspase-9 slightly increased but remained more obviously compared with the control rats (P＜0.05).MDL-28170 could attenuate the mRNA expression ofμ-calpain,Bid and AIF(P＜0.05)instead of caspase-3,caspase-8 and caspase-9(P＞0.05).ZVF could attenuate the mRNA expression of caspase-3,caspase-8 and caspase-9 instead ofμ-calpain(P＜0.05),also slightly attenuate Bid and AIF mRNA level but had no statistically significant compared with pilocarpine+saline and pilocarpine groups (P＞0.05).3.MDL-28170 attenuated the protein expression of tBid,nuclear translocation of AIF and release of cytochrome c from mitochondria(P＜0.05)but ZVF had no effect (P＞0.05).Conclusionμ-calpain mediates hippocampal neuronal PCD in LPSE-rats via caspases-independent signal pathway,mitochondrial pathway,including tBid formation from truncated Bid,AIF nuclear translocation and cytochrome c release to cytoplasm from mitochondria.SignificanceUsing a status epilepticus rat model induced by lithium-pilocarpine,we investigated and confirmed occurrence of hippocampal neuronal injury and neuronal programmed cell death after SE and involvement ofμ-calpain and caspase-3 in the different process of programmed cell death.We showed thatμ-calpain mediated neuronal programmed cell death in hippocampus via mitochondrial signal pathway including Bid and apoptosis inducing factor after SE.Furthermore,we also confirmed the neuroprotective effect ofμ-calpain inhibitor.Althoughμ-calpain inhibitor didn't decrease seizure frequence,studies onμ-calpain will help us to know more about...