The Effects And Mechanism Of COX-2 And COX-2 Inhibitors On The Epileptic Immature Rats

Background and Purpose:A large number of recalling clinical research data shows that,a very high proportion of temporal lobe epilepsy adult patients with hippocampal sclerosis can be pursued long history of epileptic seizures,particularly febrile seizures in childhood. The brain plasticity change after long epileptic seizures in early childhood can form long-term effects in the individual.Temporal lobe epilepsy with hippocampal sclerosis is closely related to the abnormal brain plasticity formation.People have concerned about the problems very much.Hippocampal axon sprouting in the loop, synaptic reconstruction is the main pathophysiological features of temporal lobe epilepsy,which may play important roles in the formation and maintenance of the temporal lobe epilepsy.In the development of epilepsy,the synapse is abnormal between cell and cell,which established pathological neural circuit.Synaptic plasticity in the hippocampus is the base of synaptic reorganization inTLE.Gliosis is resulted inTLE.The synapse consists of the presynaptic and postsynaptic terminals,which in many synapses are surrounded by gIial cell.The change of dendritic spine morphology in postsynaptic terminals induced synaptic plasticity.The nature of hippocarnpal loop was changed,which may be the foundation of chronic recurrent seizures.Cyclooxygenase2(COX-2)and its metabolites played a wide range regulation in the central nervus system,and were closely related to long-term synaptic plasticity formation and synaptic remodeling.In recent years,many studies have found that COX-2 was rapidly induced activation in the brain after prolonged epileptic seizure activity.COX-2 was widely expressed in the cerebral cortex, hippocampus,amygdala and other places and the expression was significantly increased after seizures.Some studies indicate that COX-2 inhibitors can significantly confront PTZ-induced seizure,and phenytoin joint COX-2 inhibitors can increase its anticonvulsant effect in the electric shock-induced convulsion model.It is not clear whether COX-2 is involved in the temporal lobe epilepsy with hippocampal synaptic loop reconstruction to promote the formation and development of temporal lobe epilepsy.At present,there were no further in-depth study about the anticonvulsant mechanism of COX-2 inhibitor.This study was designed to study the expression of COX-2 after epileptic seizures to see whether COX-2 inhibitors can inhibit COX-2 activity to rectify the abnormal hippocampal Synaptic remodeling in the epileptic brain caused by the change of the microenvironment and thereby attenuated the likehood of developing spontaneous recurrent seizures(SRS)after pilocarpine-induced prolonged seizures.We invisgated the effects of COX-2 inhibitor on abnormal neurogenesis and gliosis,and the cell signal transduction pathway to play the role was also studied.We also observed the effects of COX-2 inhibitor on hippocampal pyramidal neuron in vitro hippocampal tissue epileptiform discharges model through the perfusion COX-2 inhibitor to the brain slices.The alterations of voltage-dependent sodium channel,action potential(AP)and the synaptic activity were recorded by patch clamp.We studied the role and mechanism of COX-2 and its inhibitor in immature epileptic rat from the multi-angle and multi-level to provide a new control strategy for epilepsy treatment and prevention targets.Method:1.Experiment in vivo:3-week-old Wistar rats(n=120)were randomly divided into three groups: pilocarpine-induced epilepsy group(EP-Only group),Celecoxib treatment epilepsy group(EP-Celecoxib),and normal saline control group(NS).Pilocarpine-induced status epilepticus model:Rats were preteated with methylscopolamine-bromide (1mg/kg)and then single dose of pilocarpine(30mg/kg,i.p.)was administration. Seizures were scoreded by Racine's scale.Normal control rats were treated with saline instead of pilocarpine.The rats in Epilepsy-Celecoxib group were administered Celecoxib 45 minutes prior to an epileptic challenge they were fed with celecoxib(20 mg/kg,o.p.)(n=45)daily until the day of sacrifice.Also, 5-bromo-2V-deoxyuridine-5Vmonophosphate(BrdU,100 mg/kg,diluted in saline) was injected i.p.once a day for 14 consecutive days from the day of SE.(1)Animal behavior observation:comparing the difference in acute phase of status seizures and monitoring spontaneous recurrent seizure(SRS)in chronic phase from 28 to 42 days after SE;(2)Morphological Detection:The rats were sacrificed at 14 and 28 days after SE for histological analysis of COX-2,microglial activation,hippocampal cell proliferation,and migration and differentiation of BrdU-positive cells(n=10 for epilepsy-celecoxib,epilepsy-only,and normal control groups at each timing).①Nissl stain was performed to detect hippocampal neuronal damage and loss in different groups.②The immunoreactive cells in different groups were detected by immunohistochemistry and histology.The detected indexes were as followings: COX-2,OX-42 used as markers for microglia,c-fos used to map neuronal networks, BrdU and NeuN doubled stained to maker newly generated neurons,and BrdU and GFAP doubled stained to marker newly generated astroglis.(3)Western blot ddetection:Additional epileptic rats were sacrificed at 1h,1d,4d,7d,14d,and28d after SE(n=3 for each group at each timing)for western blotting.COX-2,C-fos,ERK1,ERK2 and phosphorylation of ERK1 / 2(p-ERK1 / 2) protein levels in epileptic seizures were detected at different timing.2.Expremiments in vitro:Hippocampal slices were prepared from 2wk-old Sprague-Dawley(SD)rats for whole-cell patch clamp recording.The hippocampal slice epileptic models were induced by dabbling penicillin into the slices.The alterations of the CA1 pyramidal neurons electrophysiological properties in epileptic rats after dabbling different concentrations of COX-2 inhibitor(NS-398)into the slices were analyzed by whole cell recording technology.(1)The regulations of NS-398 on pyramidal neurons membrane excitability were recorded after inducing epileptic discharge;(2)The effects of NS-398 on voltage-dependent sodium ion channels of pyramidal neurons were recorded after inducing epileptic discharge;(3)The alteration of synaptic transmission and synaptic activity caused by NS-398 were recorded after inducing epileptic discharge. Results:1.The results of experiments in vivo:(1)Observation of animal behavior:①the acute phase:seizure rate in EP-only group(89.9%)was significantly higher than in EP-Celecoxib group(55.6%)(P＜0.01); the Racine's scale in EP-only group(3.7±1.3)was significantly higher than EP-Celecoxib group(2.5±1.1)(P＜0.05);②chronic phase monitoring:Incidence of SRS in EP-Only group(50%)was significantly higher than in EP-Ceelecoxib group (30%)(p＜0.05;χ2 test);the frequency of average daily SRS in EP-group(1.9±0.58) was significantly higher than that of the EP-Celecoxib group(0.6±0.3)(p＜0.01,t test);(2)The results of morphological detection:①Nissl staining results:There were obvious damaged neurons and abnormal neuronal loss in hippocampal CA1 and CA3 and Hilus areas at 14days after pilocarpine-induced status seizures.But the EP-Ceelecoxib loss of neurons throughout the territory compared with the EP-Only group reduced(CA1 region of 48 percent vs25 percent CA3 region of 36.3 percent vs24 percent Mun Area 46 percent vs22%),;②immunohistochemical results:Ⅰ.The expression of COX-2 immune response positive cells:after pilocarpine -inducing status seizure 14 days,COX-2-positive cells in the hippocampus in the EP-only group was significantly higher than that of EP-Celecoxib group(158±18 VS 118±20)(p＜0.01).Ⅱ.microglial cell activation markers of OX-42 immunopositive cells expression: OX-42 positive cells in EP-Only group was significantly higher than that of the EP-Celecoxib group(p=0.005);Ⅲ.The results of double immunofluorescence:acute attack 28 days later,BrdU + NeuN immune double positive cells in the EP-Only group was significantly higher than that of the EP-Celecoxib group(36±4 Vs 22±3);at the same time BrdU+ GFAP double stained positive cells in the EP-Only group was significantly higher than that of the EP-Celecoxib group(26±3 Vs 14±2)EP-Only group in Hilus. COX-2 inhibitors can inhibit the production of glial cells;(3)Western blotting results:①The expression of COX-2 protein was low leveling normal control group,and the COX-2 protein was expressed in a time-dependent manner after SE.It peaked at 1day after SE and declined thereafter,but it was still higher than the normal control group at 28 days after SE.Celecoxib significantly attenuated the COX-2 expression in the rat brain over the latent period after SE.At 4 days after SE,the COX-2 expression was 1.3 times in the EP-Only group compared with the EP-Celecoxib group,and the former is 1.5 times than the latter at 14 days after SE;②C-fos protein concentrations in the 1h,1d,4d,7d and 14d in the EP-only group were significant higher that of EP-Celecoxib group.The c-fos protein expression peaked 1d and then gradually declined.The concentration of c-fos protein declined to the normal control level at 28d after SE,and there was no significant difference comparing with the control group(P＞0.05);③MAPK / ERK signaling system and the activities of the expression:the phosphorylation of ERK1 / 2 levels increased rapidly at 1hour after pilocarpine induced seizure,it peaked at 1d(10-times compared to the normal group),the phosphorylation of ERK1 / 2 expression was significantly lower in EP-Celecoxib group than the EP-Only group(p＜0.01)but still higher compared with the control group(p＜0.01).The expressions of Non-phosphorylation of ERK1 and ERK2 in hippocampal were intensive than phosphorylation of ERK1 / 2,but there were on significant increase after seizures(p＞0.05).2.The results of the experiments in vitro:The effects of NS-398 CA1 pyramidal neurons electrophysiological characteristics in penicillin induced epileptic hippocampal slices:(1)The impact of NS-398 on the voltage-dependent sodium current:NS-398 can reduce the current rate and extent the no-answer period of the voltage-dependent sodium channel to decreasing the open frequency of sodium channel by extending inactivation recovery phase from deactivation,and neuronal action potential frequency of issuance corresponding declined;(2)The impact of NS-398 on the action potential:COX-2 inhibitor NS-398 inhibited the magnitude and frequency of action potential,extended the action potential latency,which eventually led to pyramidal neurons to delay the discharge of action potentials and decrease the release of neurotransmitters;(3)The effect of NS-398 on synaptic activity and transmitter release:NS-398 significantly inhibited the frequency of sEPSCs ofthe CA1 pyramidal neurons in epileptic rat hippocampal slices,but there was no obvious impact on the rate of decay time of sEPSCs.Meanwhile NS-398 significantly enhanced the frequency and significantly prolonged the decay time of sIPSCs,but little impact on the current rate. Conclusion:1.The activation of COX-2 can be induced rapidly after seizures,and COX-2 inhibitor attenuated the likelihood of developing spontaneous recurrent seizures after pilocarpine-induced prolonged seizure.2.During the latend period,COX-2 inhibitor prevented neuronal death and microglia activation in the hilus and CA1 and inhibited the generation of ectopic granule cells in the hilus and new glia in CA1.3.COX-2 inhibitors can inhibit the activation of MAPK / ERK signaling pathway and its downstream signaling molecule C-fos activation to inhibit the abnormal cell proliferation and differention after prolonged seizures.4.COX-2 inhibitors can reduce the membrane excitability of pyramidal neurons in hippocampus,extend the inactivation time of voltage dependent sodium ion channel,reduce the discharge of action potential and reduce the release of sEPSCs and reinforce the release of sIPSCs to reduce the release of excited neurotransmitters.5.These findings raise the evidence of COX-2 induction to act importantly on epileptogenesis and suggest a potential therapeutic role for COX-2 inhibitors in chronic epilepsy.