Sodium Valproate Monotherapy On Neutrophil Function In Children With Epilepsy

Epilepsy,with a morbidity of 4.8‰-11.2‰,is a common nervous system disease. Valproate acid(VPA),a conventional first-line antiepileptic drug,is generally used in clinics,especially in pedietrics.Nearly 1/3 newly diagnosed epileptic patients are given VPA treatment up to now.Besides,VPA are used to treat bilateral mood disorder and some tumors,and are effective in preventing and treating migraine.An increasing number of researches focused on the effects of epilepsy and antiepileptic drugs(AEDs) on immune system since Walker proposed the standpoint that innune system may play an important role in the phatophysiological of epilepsy. Chronic VPA monotherapy may influence many human systems including immune system.In clinics,we found that epileptic children with VPA monotherapy were invulnerable to infection which may be result from their depressed immune system function.Previous studies showed that VPA monotherapy could result in transient decrease of serum albumin in epileptic children.Besides,VPA inhibits the production of proinflammatory cytokines like IL-6,IL-2 and TNF-a and inhibits the activation of nuclear factor kappa B(NF-KB) which is an ubiquitous and important transcription factor for genes that encode the kappa light chain of immunoglubulin and proinflammatory cytokines,such as IL-1,IL-6,IL-8 and TNF-a.But previous studies were mostly focused on the effects of VPA on humoral immunity and lymphocyte,there were no researches about the effects of VPA on neutrophilic function up to now.Several researches showed that VPA could mediate the chemataxis and phagocytosis activity through attenuate the Phospholipid Signaling in the model of Social aoeba Dictyostelium discoideum which is a useful model for investigating neutrophil chemotaxis and the molecular mechanisms that regulate this process because of the similarities in the way in which both celltypes move and respond to che motactic signals.Dictyostelium has been particularly useful for the study of chemotaxis and cell motility,differentiation and morphogenesis.Meanwhile,VPA could induce imbalance of the status of oxidative and antioxidative,and increase the production of neurotransmitter GABA in mammalian animal neurons to reduce seizures.Neutrophlil is an important ingredient of primary immunity,and play an important role in mediating inflammatory reaction and immunological response.Chemataxis and phagocytosis activity are the basic and primary function of neutrophils which is similar to that of the eukaryote.So,in our study,we proposed the question that can VPA mediate immune system through mediate the chemataxis and phagocytosis activity of neutrophils? Before our study,we have established an easy and quick way to valuate the neutrophilic function through which we can detect chemataxis and phagocytosis activity of neutrophil simultaneouly.In our study,we tried to illuminate the feature and mechanisms of the effect of VPA on the neutrophlic function through detect the neutrophilic function before and after VPA monotherapy,and studied the immune status of epileptic chileren under VPA monotherapy with a new angel.OBJECTIVES1.To investigate the changes of neutrophilic function in epileptic children before and after VPA monotherapy.2.To investigate the features and mechanisms of the effect of VPA on the neutrophlic function3.To instruct the clinical use of VPAMETHODSThere are two parts of this study:PartⅠ:in vivo PartⅡ:in vitro1.ObjectAccording to the diagnostic criteria of ILAE 1989,we selected children whose both clinical manifestation and EEG result were accord with the diagnostic criteria in the out-patient clinics,and divided them into two groups:general seizures group and focal seizures group.Including criteria: ①Clinical menisfection and EEG results are accord with the diagnotic criteria of epilepsy.②Not take any antiepileptic drug before be diagnosed as epileptic patients③Be suitable for VPA therapy④VPA monotherapy without other chronic disease and associated drug treatment⑤In vitro,select healthy children in the out-patient clinics whose age and gender are matched with the epileptic children.Excluding criteria①Seizures,inflammation or stress state one week before detecting neutrophilic function②Take immunomodulating drugs or ACTH six monthes before VPA monotherapy or before health examination③Change to other antiepileptic drugs monotherapy or therapeutic alliance during VPA monotherapy④Immune deficiency or changed neutrophilic function⑤Have brain disease before,such as encephalitis,meningitis,brain development malformation,brain tumors or mental disorder.2.MethodIn vivo,we selected the epileptic children acording to the including and excluding criteria and detected their neutrophilic function before and after VPA monotherapy.In the first stage of in vitro study,we selected the healthy children whose age and gender matched the epileptic children in the out-patient clinics to draw blood,and gave whole blood culture,addin sufficient quantum VPA to get 0,50,100,150,200μg/ml VPA concentration,and the 0μg/ml group as the control group.The whole bloods were maintained at 37℃in humidified 5%CO2.Detected the neutrophilic function before and after 6h,18h and 24h culture respectively;in the second stage of in vitro study, we adopted 0,50 and 200μg/ml VPA concentration,divided each sample into two groups 6 hours after whole blood culture:one group stimulated by PMA,another gave NS only.detected the CD64 and MPO levels before and after 6h whole blood culture respectively and statistically analyse the data with SPSS 13.0. 3.Statistical analysisThe measurement data were showed in the form of X~-±S.The neutrophilic function before and after VPA monotherapy were analysed with paired sample T test.The neutrophilic function between general seizures and focal seizures were analysed with independent sample T test.The neutrophilic function,levels of CD64 and levels of MPO between each VPA concentration group and the control group were analysed with paired sample T test.Either between the 200μg/ml group and the 50μg/ml group.when P＜0.05,the disparity between the two group are have statistical significance.we analysed our data with SPSS 13.0.RESULT1.Genaral informationDuring the study,we included 64 epileptic patients according the including and excluding criteria,within which there were 34boys and 30 girls;39 general seizures and 25 focal seizures.Their age ranged from 1 year old to 12 years old,the average age is 4.5 year old before VPA monotherapy and that were 1.5 year old to 13 and 13 years old after VPA monotherapy respectively.The median age were 3.17 and 3.75 years old before and after VPA monotheraoy respectively.The mean follow up period is 6 monthes.2.Effects of VPA monotherapy on neutrophilic functionThe auto-active rate of neutrophils are increased after VPA monotherapy,and partly showed in the form of double hump on the flow cytometry.The auto-active rate of neutrophils before and after VPA monotherapy are(8.84±10.3)%and(15.0±11.6) %respectively,the difference have statistical significance.The stimulate index before and afetr VPA monotherapy are 444.4±306.2 and 350.7±226.6respectively, the difference have statistical significance,which hint that the reserved function of neutrophil are decreased.In this study,the difference of neutrophilic function between general seizures and focal seizures have no statistical significance,So the effect of VPA monotherapy on neutrophilic function have no relation with the seizure types.3.Effects of VPA monotherapy on neutrophilic function in vitro The auto-active rate of neutrophils are increased according with the increase of VPA concentration in a dose-depending way.and the differences in auto-active rate of neutrophils of each VPA concentration group and the control group have statistical significance.But neutrophils is fragile in votro,many neutrophils died after 18h and 24h whole blood culture,so the auto-active rate decreased with time prolonged.The stimulate index decreased with the increase of VPA concentration in a dose-depending way.Though at 6h timepoint under 50μg/ml VPA concentration the stimulate index increased lightly,it degrade significantly after 24h culture.4.Effects of VPA monotherapy on the level of CD64 and MPO in vitroThere were no statistical significance in the level of CD64 between each VPA concentration group and the control group.Either between the 50μg/ml group and the 200μg/ml group.The level of MPO decreased with the increase of VPA concentration in a dose-depending way.The difference between each VPA concentration group and the control group had statistical significance,Either between the 50μg/ml group and the 200μg/ml group,which suggests that the oxidative activity are impaired after VPA monotherapy.DISCUSSION1.Generally,though neutrophilic function enhanced after VPA monotherapy,it degrades strikingly once the body are stimulate by microorganisms or stress, which suggests that neutrophilic reserved function degrades.2.Anti-effective ability in epileptic children treated with VPA degrades.3.Effects of VPA on neutrophilic function is time-depending,neutrophilic function degrades with time prolonged.4.Effects of VPA on neutrophilc function is dose-depending,neutrophilic function degrades with dose inceresed,which suggest that VPA blood concentration should be controlled to a low level under the premise that seizures were controlled well in clinics.5.VPA mediate neutrophilic function through degrading its oxidative activity,but we need further study to make sure if VPA can mediate its chemotaxis activity.6.We get the result from common epiliptic children only treated with VPA,this results could not be generalized to epileptic children with other chronic disease and associated drug treatmentConclusion1.Neutrophilic reserved function in epileptic chilerdren treated with VPA degrades3.Effects of VPA on neutrophils is negatively dose -depending4.Anti-effective ability in epileptic children treated with VPA may degrades.