Empirical Study On FK506 Ameliorating Coures Of Epilepsy And Ability Of Learning And Memory By Inhibiting Calcineurin

BackgroundTemporal lobe epilepsy(TLE) is a common form of epilepsy in adults,Morbidity rate for epilepsy is 5‰～6‰according epidemiologic survey.It is harm on people's healthy and life and has high fatality rate.About 20%patients with temporal lobe epilepsy has been diagnosed as refractory seizure,which is ralated to hippocampus stiffen in temporal lobe.The studies from epileptic patient and animal model showed that temporal lobe seizures may induce local or diffuse neurodegeneration depending on intensity and duration of seizures.The change,if irreversible,may lead to permanent neurological impairments and susceptibility to spontaneous seizures.Seizures can result in increase in Ca²⁺ release and Calmodulin(CaN) activity,subsequently initiating excitotoxic processes leading to mitochondrial dysfunction and neuronal cell death.Calmodulin,also called calcineurin(CaN),is a member of proteinphosphatase of Serine/threonine family.CaN is a calcium/calmodulin-stimulated phosphatase,enriched in neural tissue.CaN-mediated dephosphorylation is an important modulatory factor in many cellular processes,including development of learning and memory,regulation of long-term potentiation(LTP) and induction of apoptosis.Additionally,CaN may depress the activity of the GABA receptor,an important inhibitory receptor in the brain.An immunosuppressive agent,FK506,has been used in clinical settings for the prevention of allograft rejection,which orchestrates its immunosuppressive action by binding to FK506 binding protein 12(FKBP12).Recent studies in vitro and vivo have demonstrated that FK506 not only induces some neurological diseases such as focal cerebral ischemia,but also plays a role in neuroprotection and neurotrophy.In kainate-induced seizure,FK506 was shown to protect neuron through preventing the sprouting of mossy fibres in the hippocampus.In middle cerebral artery occlusion model, cortical damage was remarkably reduced by intravenous injection of FK506.Despite the protective effect of FK506 on neuron was observed in experimental cerebral ischemia and seizure model,the mechanism by which FK506 targets neuron remains unclear.Therefore,we explore the questions that whether and why treatment with FK506 could ameliorate the course of ongoing seizures and improve the ability of learning and memory of rats.The results showed that there was an increase in CaN activity at SE2h and CaN protein express at SE24h,which all result in decrease in frequency and amplitude of IPSC and GABA_AR dephosphorylation and down-regulation, suggesting increase of susceptibility to spontaneous seizures and form of refractory epilepsy.FK506 could control refractory epilepsy and prevent neuron from damage by inhibiting CaN activity,CaN protein express and GABA_AR dephosphorylation.FK506 plays important role in treatment of refractory epilepsyPartâ… Study of EEG and pathology of Kainic acid model of temporal lobe epilepsyObjectiveTo investigate the alteration of behavior,electrophysiology and pathology of KA model of temporal lobe epilepsy,and so to decipher seizure-dependent changes in neuronal injury in rat hippocampus after temporal lobe epilepsy induced by Kainic acid (KA) injection.Method1.Kainic acid(0.4ug/ul,Sigma,France) was intracerebroventricular injection by stereotactic operation to make modle of temporal lobe seizures.Rats were randomly divided into control group,SE2h(status epileptics,SE) group,7d(SE7d) group and 45d (SE7d) group after KA injection. 2.To observe seizure behavior according to a modified Racine scale by video recording and EEG changes of cortical layer using biological function recorder among 24h,at 7d,45d after SE induced by KA.3.To evaluate pathology features of hippocampal neurons of each group by Nissl staining and number of mossy fiber sprouting(MFS) by Timm staining.4.To detect GFAP immunoreactive positive cells in hippocampus by immunohistochemistry.Result1.Behavioral episodes induced by KA injection showed a typical process of seizure from a gradual increase of intensity to status epilepticus,and subsequently abated, however,seizure was still seen at 15 day after KA injection.The mortality rate of these rats was 10%(6/60).EEG showed accumulated spike waves in acute phase,normal during the latency phase and showed epileptic waves during the chronic phase.2.The pathologic investigation using Nissl staining showed nomal neuron at control group,but the hippocampal neuronal damage at SE2h group,SE7d group and SE45d group.The hippocampal neuronal damage became serious as time going.3.Timm staining showed little MFS at control group and SE2h group.However, Timm staining showed a little MFS at SE7d group,and much more increase at SE45d group.4.Immunohistochemistry showed that prolonged seizures induce GFAP immunoreactivity enhanced in the hippocampus after seizures,indicating astroglisis.Conclusion1.Kainic acid injected into the right intracerebroventricular can successfully make modle of temporal lobe seizures,which is safe,convenient and feasible.Epilepsy induced by KA develops in 3 phase according to alteration of behavior and EEG:acute phase(status epilepticus),latency phase and chronic phase(spontaneous recurrent seizures).2.Epileptic seizures induced by KA can cause neuronal damage,MFS and astroglisis in the hippocampus of rats. Partâ…¡Experimental study on FK506 ameliorating the course of temporal lobe epilepsy and improving ability of learning and memoryObjectiveTo investigate an immunosuppressant,FK506,whether or not can ameliorate the course of temporal lobe epilepsy and improve ability of learning and memory and protect from ultramicro demage caused by seizures.Method1.KA was injected into the right intracerebroventricular to make modle of temporal lobe seizures.The seizure activity of rats induced by KA was scored for 6h using a modified Racine scale.Rats were randomly divided into control group,SE2h(status epileptics,SE) group,FK506 before KA group and FK506 after KA group.2.To observe seizure behavior according to a modified Racine scale by video recording and the effect of FK506 on seizure behavior.3.To evaluate cognitive function in every experiment subgroups by behavioral tests and the effect of FK506 on cognitive function.4.To evaluate pathology features of hippocampal neurons of each group by Nissl staining and the effect of FK506 on pathology features.5.Mitochondrial ultrastruture damage in rats with FK506 or without FK506 was evaluated by electron microscope.Result1.Behavioral episodes induced by KA injection showed a typical process of seizure from a gradual increase of intensity to status epilepticus,and subsequently abated. Compared with KA group,the lasting time of maximal seizure and times of seizures showed significant decrease in FK506 before KA group and FK506 after KA group.2.Morris water maze showed the cognitive function of KA group became worse (P＜0.05),when compared with control or FK506 before KA group or FK506 after KA group. 3.No extensive destruction was noted in the dentate gyrus,CA1 and CA3 subfields of hippocampus of rats of control group.Compared to control group,KA injection resulted in a loss of neuron in pyramidal cells of hippocampus,especially in the CA1, CA3 area.Nissle's staining showed shrinkage of neuron as triangle or round and condensation of nucleus.It has been observed in some neurons that cytoplasm appears heavy staining and nucleolus disappears with pyknotic nucleus(all P＜0.01).The rats with FK506 before or after KA injection revealed lower pathological scores than KA group rats(all p＜0.05).4.Compared to control group,mitochondrial ultrastructure showed damaged, mitochondrial swelling was accompanied by clearing of matrix density and disruption of membrane integrity.The most severe damage of mitochondria was vacuolar plus rupture of inner and outer mitochondrial membranes.(all P＜0.01) However,the KA-induced loss of neuron in CA1,CA3 layer and mitochondrial ultrastructure damages were significantly reduced by treatment with FK506(all p＜0.05).Conclusion1.Behavioral episodes induced by KA injection showed a typical process of seizure.Animals treated with FK506 before or after KA appeared to significantly decrease in susceptibility to seizures.Compared to KA group,the times of seizures were markedly lower and lasting times of maximal intensity symptoms was also shorter in FK506 before or after KA group.It suggests that FK506 could inhibit temporal lobe epilepsy induced by KA.2.The learning and memory markedly decreased in rats induced by KA.FK506 could shorten times to find platform,increase swimming distance and frequecy in the target zon,which indicates that FK506 could improve learning and memory.3.FK506 could reverse shrinkage,necrosis and damage of mitochondria of hippocampal neuron induced by KA.It suggests that FK506 plays a role in protecting hippocampal neurodegeneration induced by KA. Partâ…¢Experimental study on FK506 meliorating epilepsy by up-regulation of GABAergic synaptic transmissionObjectiveTo evaluate changes of IPSC(inhibitory postsynaptic currents) using whole-cell patch-clamp recordings after seizure induced by KA;and to observe dynamic changes of CaN activity,CaN protein expression and GABA_A receptor(GABA_AR);and also to reseach on anti-epilepsy mechanisms of FK506.Method1.KA was injected into the right intracerebroventricular to make modle of temporal lobe seizures.2.Whole-cell patch-clamp recordings from CA3 pyramidal neurons of acutely removed hippocampal slices.Slices were incubated,Inhibitory postsynapti curret(IPSC) frequency and amplitude in CA3 pyramidal neurons were observed.Both application of CaN inhibitor FK506 and Rapamycin were been used to understand their effects on IPSC frequency and amplitude.3.Hippocampus CA3 Slices were prepared in sham group,KA goup,FK506 before KA group and FK506 after KA group.Both basal and Mn²⁺ maximally stimulated CaN activity were measured using this assay procedure in area CA3 at 2h after maximal intensity of seizure(SE).CaN protein levels after 2,6,12,24h were measured using Western blot analysis respectively.4.By Westernblot analysis,we measured the phosphorylation state of GABA_AR 2/3 subunits after KA-induced seizures using anti-phospho serine/threonine antibodies against immunoprecipitated GABA_AR 2/3 subunit protein and effect of FK506 on it.Results1.Frequency and amplitude of IPSC in CA3 pyramidal neurons were decreased on 2h after SE.In CA3 pyramidal neurons,both IPSC frequency and amplitude were significantly decreased in slices from KA-treated rats compared with those from sham animals.IPSC were completely blocked by bath application of the GABA_AR antagonist bicuculline methiodide(20μM),which Confirming that IPSCs were mediated by GABA_ARs.The mean IPSC frequency in the KA group was decreased by 53.4±3.7%(1.72±0.1 Hz for sham groups and 0.80±5.6 Hz for KA-treated rats;n=5 cells per group;p＜0.01),and the mean amplitude was decreased by 38.4±7.1%(19.8±2.1 pA for sham and 12.2±1.2 pA for KA-treated rats;p＜0.05;n=5 cells per group).But compared with KA group,the mean IPSC frequency of FK506 increased 113.8±21.1%(from KA group 0.80±0.5 Hz to FK506 group 1.71±0.11Hz,p＜0.01,n=5 cells per group),the mean IPSC amplitude of FK506 increased 48.4±4.1%(from KA group 12.2±1.2pA to FK506 group 18.1±0.2 pA,p＜0.05;n=5 cells per group).However,compared with KA group,the mean IPSC frequency or amplitude of Rapamycin(non CaN inhibitor) had no changed(from KA group 0.80±0.5 Hz to Rapamycin group 0.91±0.03 Hz or from KA group 12.2±1.2 pA to Rapamycin group 11.5±0.4 pA,n=5 cells per group,all p＞0.05),which indicated FK506 increased frequency and amplitude of IPSC and GABAergic synaptic inhibition by inhibitting CaN.2.FK506 decreased CaN activity.CaN activity is elevated in hippocampal slices after seizure compared with shaml levels.Slices from the KA-treated group exhibited a significant increase in CaN phosphatase activity in both basal and maximal conditions. Compared with the KA-treated group(28.35±0.82 ug/ml),the average basal activity in sham group slices(18.11±1.25ug/ml,n=5) increased 56.54±3.25%(n=5;p＜0.01). Compared with the KA-treated group(42.12±5.82 ug/ml),the average maximal activity in shaml slices(25.92±3.85 ug/ml) increased 62.5±1.05%(n=5;p＜0.01).But compared with the KA-treated group,CaN average basal activity and average maximal activity exhibited a significant decrease in FK506 before or after KA groups(FK506 before KA:basal:18.25±1.15ug/ml,n=5,p＜0.01;Max:25.82±2.91ug/ml,n=5,p＜0.01;FK506 after KA:basal:21.23±2.53 ug/ml,n=5,p＜0.01;Max:26.17±1.54 ug/ml,n=5,p＜0.01).3.Effect of FK506 on CaN protein expression:CaN protein expression was not changed until SE24 h.FK506 decreasesd CaN protein expression at SE24h(n=5;p＜0.05).4.FK506 inhibits serine/threonine residues of GABA_AR2/3 subunits dephosphorylated after KA-induced seizures.After SE 2h,there was a significant decrease in the level of phosphorylation of serine/threonine residues in GABA_AR 2/3 subunits by 38.2±4.8%of the sham with no alteration in total 2/3 subunit protein(n=5;p＜0.01),After with treatment FK506 before of after KA,there was a significant increase in the level of phosphorylation of serine/threonine residues in GABA_AR 2/3 subunits by 62.7±3.8%, 59.3±2.5%of the sham with no alteration in total 2/3 subunit protein(n=5;p＜0.05).Conclusion1.KA-induced seizures result in not only acute increases of CaN activity and GABA_AR dephosphorylation in the hippocampus,but also decrease basal synaptic inhibition of hippocampal CA3 pyramidal neurons,which bring out intractable epilepsy and severe injury of neuron.CaN inhibition FK506 partially reverses the effects of KA-induced seizures by increasing basal synaptic inhibition and up-regulation GABAergic inhibition,improves learning and memory of rat and protects hippocampal neurons.2.CaN protein expression showed no change until 24h after seizure,which indicated CaN protein expression could be related with chronic epilepsy via increasing susceptibility to seizures.FK506 could decrease CaN protein expression at 24h after seizure.Our data indicate that FK506 and/or its analogs may have clinical potential in the treatment of refractory perinatal seizur. To investigate the effects of FK506 on the alteration of behavior,electrophysiology, ability of learning and memory and pathology in the temporal lobe seizures model of rat induced by KA intracerebroventricular injection,and understand the effects of FK506 on frequency and amplitude of inhibitory postsynaptic currents(IPSC) from CA3 pyramidal neurons using whole-cell patch-clamp recordings.To our knowledge,it is the first research that CaN is involved in temporal lobe seizure in vivo and vitro.The results showed there was an increase in CaN activity at SE2h and CaN protein express at SE24h, which all result in decrease in frequency and amplitude of IPSC and GABA_AR dephosphorylation and down-regulation,suggesting increase of susceptibility to spontaneous seizures and form of refractory epilepsy.FK506 could control refractory epilepsy and prevent neuron from damage by inhibiting CaN activity,CaN protein express and GABA_AR dephosphorylation.FK506 plays important role in treatment of refractory epilepsy...