Pentoxifylline Ameliorates Function Of Dopaminergic Neurons In SN-CPu Of Lithium-pilocarpine Induced Epileptic Rats

Epilepsy is a chornic disorder which characterized predominantly bytransient or persistent brain dysfunction resulting from repeated abnormalspontaneous discharge of neurons. Studies found that ES has a closelyrelationship with dopaminergic nervous system. Researches showed that thedensity of dopamine receptors has significantly changed in epilepsy rats. Thedensity of dopamine (DA) and its metabolite homovanillic acid (HVA) hasdecreased in cerebrospinal fluid of epilepsy patients and the density of DAincreased after antiepileptic treatment, which suggested that dopaminergicnervous system may be affected in the process of epileptic seizures (ES). Atpresent, the mechanism of ES is not clear. Some researches suggested adefinite role for the production of oxygen free radicals in the excessivedischarge of neurons in the brain of status epilepticus (SE) rat. Freitas et al.also confirmed that the active oxygen free radicals affect of neurons ofepilepsy rat. Therefore inhibition the over-excited neurochemical pathwayscan reduce the production of oxygen free radicals, relieved neuron injury andameliorate related symptoms of the epileptic seizure, which is probably one ofthe methods of epilepsy treatment.Pentoxifylline (PTX) is a nonspecific phosphodiesterase inhibitor, has astrong antioxidant effect, and can remove oxygen free radicals from the body.This drug was initially used in the treatment of peripheral circulatory diseasesand respiratory disorders. Recent studies have shown that PTX can be used inthe treatment of neurological disorders, such as ischemic brain injury andstroke. In addition, Geanne et al. found that PTX can improve the learningacquisition and memory of rats which hippocampus damaged by glutamate,another study found that intraperitoneal pretreatment of animals with PTXsignificantly lower the seizures rate and decreased the severity of seizures of rats induced by lithium-pilocarpine (Li-Pc), suggesting the antiepileptic andneuroprotective effects of PTX. The DA neurons in SN-CPu of rats areaffected in the process of ES, while there is no study shows whether PTX canameliorate the damages.Epileptic seizure is often sudden attack outside the hospital. The injuryseverity of brain neurons is related to the duration of epileptic seizures, longtime of epilepsy seizures will endanger the lives of patients, timely andeffectively terminated seizures can reduce the damage to brain neurons whichhas a important role to promote the quality of patient’life. At present somestudies showed that it is effective to treat ES through intranasal administration.Intranasal administration is a quick and convenient way to control suddenonset of epilepsy. Intranasal administration of drugs is convenient, which hasbeen become one of the preferred methods in recent year. However, it is stillunclear whether intranasal administration of PTX has antiepileptic effect. Theepilepsy rat induced by Li-Pc showed hippocampus neuron death and mossyfiber sprouting (MFS) which similar with the pathological and clinical featuresof humanity temporal lobe epilepsy (TLE), it is a ideal model of TLE andsuitable for exploring the mechanism of epilepsy and screening antiepilepticdrugs (AEDs).Therefore, epileptic rats induced by Li-Pc were used in the present study.Real-time reverse transcription-quantiative PCR (RT-qPCR), Western blot andliquid chromatography-mass spectrometry (LC-MS) were used in this study toobserve the related indexes which can reflect the function of the DA system.To study the effect of intraperitoneal and intranasal pretreatment of PTX onthe DA neurons in SN-CPu of rats, the result is hoped to provide experimentalevidence for the prevention and treatment of epilepsy.Objective: To observe the influence of PTX on dopaminergic neurons inSN and CPu of ES rats and analyze the efficacy of PTX treatment byintraperitoneal injection and intranasal administration, it is hoped that theresults can provide some experiment basis for ES and its treatment.Methods: The male Wistar rats (300g) were randomly divided into intraperitoneal injection and intranasal administration groups.(1)Intraperitoneal injection group:50rats were randomly divided into5groupsnamed control group (Con.ip), lithium chloride group (Li.ip), intraperitonealadiministion of pentoxifylline control group (PTX.ip), epileptic seizure group(ES.ip), epileptic seizure supplemented with intraperitoneal adiministion ofpentoxifylline group (ES+PTX.ip). The animals in Con.ip and PTX.ip groupsreceived saline (127mg/kg) alone, respectively. The animals in Li.ip groupreceived Li (127mg/kg) alone. ES was induced in other two groups byadministering an aqueous (saline) solution of lithium chloride (127mg/kg)intraperitoneally, followed (20hours later) by pilocarpine (15mg/kg)intraperitoneall. PTX.ip and ES+PTX.ip groups were administerstered PTX(60mg/kg) intraperitoneally in30min before pilocarpine injection. Eachgroup has10rats (5rats were used for RT-qPCR and LC-MS, the others wereused for Western blot).(2) Intranasal administration group:50rats wererandomly divided into5groups named control group (Con.in), lithiumchloride group (Li.in), intranasal adiministion of pentoxifylline control group(PTX.in), epileptic seizure group (ES.in), epileptic seizure supplemented withintranasal adiministion of pentoxifylline group (ES+PTX.in). The animals inCon.in and PTX.in group received saline (127mg/kg) alone, respectively. Theanimals in Li.in group received Li (127mg/kg) alone. ES was induced in othertwo groups by administering an aqueous (saline) solution of lithium chloride(127mg/kg) intraperitoneally, followed (20hours later) by pilocarpine(15mg/kg) intraperitoneall. ES+PTX.in and PTX.in groups wereadministerstered PTX (60mg/kg) intranasally in30min before pilocarpineinjection. Each group has10rats (5rats were used for RT-qPCR and LC-MS,the others were used for Western blot).At the eighth day after the success of induced ES, observe the influenceof PTX on the expressions of tyrosine hydroxylase (TH) mRNA，dopaminetransporter (DAT) mRNA，TH and DAT in the Substantia nigra (SN)-Caudateputamen (CPu) of ES rats by RT-qPCR and Western blot and detect the effectson DA and its metabolites HVA and dihydroxyphenylacetic acid (DOPAC) in intraperitoneal and intranasal pretreatment of PTX in CPu of ES rats byLC-MS.Results:1The effects on dopaminergic neurons in SN-CPu of rats by giving PTXintraperitoneally1) Results of RT-qPCRBy RT-qPCR, we tested changes of the expressions of TH mRNA andDAT mRNA in SN of rats. Compared with Con.ip group, the relativeexpressions of TH mRNA and DAT mRNA in SN of rats in PTX.ip grouprespectively increased by19%(P<0.01) and16%(P<0.01); Compared withCon.ip group, the relative expressions of TH mRNA and DAT mRNA in SN ofrats in ES.ip group respectively decreased by28%(P<0.01) and26%(P<0.01); Compared with ES.ip group, the relative expressions of TH mRNAand DAT mRNA in SN of rats in ES+PTX.ip group respectively increased by30%(P<0.01) and34%(P<0.01) and close to normal rats.2) Results of immunoblottingEffects on the expressions of TH in intraperitoneal injection group:Compared with Con.ip group, the relative expressions of TH in CPu and SNof rats in PTX.ip group respectively increased by31%(P<0.05) and21%(P<0.05); Compared with Con.ip group, the relative expressions of TH in CPuand SN of rats respectively decreased by48%(P<0.01) and45%(P<0.01);Compared with ES.ip group, the relative expressions of TH in SN and CPu ofrats in ES+PTX.ip group respectively increased by91%(P<0.01) and89%(P<0.05) and close to normal rats.Effects on the expressions of DAT in intraperitoneal injection group:Compared with Con.ip group, the relative expressions of Glycosylated-DAT(Glyco-DAT) in SN and CPu of rats in PTX.ip group respectively increasedby26%(P<0.05) and9%(P<0.05); Compared with Con.ip group, the relativeexpressions of Glyco-DAT in SN and CPu of rats in ES.ip group respectivelydecreased by54%(P<0.01) and46%(P<0.01); Compared with ES.ip group,the relative expressions of Glyco-DAT in SN and CPu of rats in ES+PTX.ip group respectively increased by114%(P<0.01) and73%(P<0.01), close tonormal rats. The relative expressions of Non-Glycosylated-DAT(Non-Glyco-DAT) in rats of all groups did not change significantly.3) Results of LC-MSAnalyzed the results of LC-MS found: Compared with Con.ip group, thedensity of DA, DOPAC and HVA in CPu of rats in PTX.ip group respectivelyincreased by11%(P<0.05),14%(P<0.05) and17%(P<0.05); Compared withCon.ip group, the density of DA, DOPAC and HVA in CPu of rats in ES.ipgroup respectively decreased by41%(P<0.01),45%(P<0.01) and47%(P<0.01); Compared with ES.ip group, the density of DA, DOPAC and HVAin CPu of rats in ES+PTX.ip group respectively increased by65%(P<0.01),56%(P<0.01) and53%(P<0.01) and the density of DA in CPu of rats inES+PTX.ip group is close to the density of DA in CPu of rats in Con.ip group,but the density of DOPAC and HVA in CPu of rats in ES+PTX.ip group is stillsignificantly lower than mormal rats.Compared with Con.ip group, the relative values of (DOPAC+HVA)/DA,DOPAC/DA and HVA/DA of rats in ES+PTX.ip group are respectivelydecreased by9%(P<0.05),8%(P<0.05) and12%(P<0.05). The relativevalues of (DOPAC+HVA)/DA, DOPAC/DA and HVA/DA of rats in othergroups did not show significant differences.2The effects on dopaminergic neurons in SN and CPu of rats by giving PTXintranasally1) Results of RT-qPCRCompared with Con.in group, the relative expressions of TH mRNA andDAT mRNA in SN of rats in PTX.in group respectively increased by18%(P<0.05) and21%(P<0.01); Compared with Con.in group, the relativeexpressions of TH mRNA and DAT mRNA in SN of rats in ES.in grouprespectively decreased by28%(P<0.01) and32%(P<0.01); Compared withES.in group, the relative expressions of TH mRNA and DAT mRNA in SN ofrats in ES+PTX.in group respectively increased by35%(P<0.01) and48%(P<0.01) and close to normal rats. 2) Results of immunoblottingEffects on the expressions of TH by giving PTX in intranasaladministration: Compared with Con.in group, the relative expressions of THin SN and CPu of rats in PTX.in group respectively increased by21%(P<0.05)and18%(P<0.05); Compared with Con.in group, the relative expressions ofTH in SN and CPu of rats in ES.in group respectively decreased by59%(P<0.01) and51%(P<0.01); Compared with ES.in group, the relativeexpressions of TH in SN and CPu of rats in ES+PTX.in group respectivelyincreased and close to normal rats.Effects on the expressions of DAT by giving PTX in intranasaladministration: Compared with Con.in group, the relative expressions ofGlyco-DAT in SN and CPu of rats in PTX.in group respectively increased by43%(P<0.01) and17%(P<0.01); Compared with Con.in group, the relativeexpressions of Glyco-DAT in SN and CPu of rats in ES.in group respectivelydecreased by57%(P<0.01) and53%(P<0.01); Compared with ES.in group,the relative expressions of Glyco-DAT in SN and CPu of rats in ES+PTX.ingroup respectively increased by138%(P<0.01) and117%(P<0.01), close tonormal rats. The relative expressions of Non-Glycosylated-DAT(Non-Glyco-DAT) in rats of all groups did not change significantly.3) LC-MSCompared with Con.in group, the density of DA, DOPAC and HVA inCPu of rats in PTX.in group respectively increased by12%(P<0.05),15%(P<0.05) and13%(P<0.01); Compared with Con.in group the density of DA,DOPAC and HVA in CPu of rats in ES.in group respectively decreased by43%(P<0.01),36%(P<0.01) and45%(P<0.01). After giving PTXintranasally, the density of DA, DOPAC and HVA in CPu of rats inES+PTX.in group is close to the density of DA, DOPAC and HVA in CPu ofrats in Con.in group and compared with ES.in group the density of DA,DOPAC and HVA in CPu of rats in ES+PTX.in group respectively increasedby68%(P<0.01),40%(P<0.01) and69%(P<0.01).The relative values of(DOPAC+HVA)/DA, DOPAC/DA and HVA/DA in rats of all groups did not change significantly.3The observation targets of control groups and lithium chloride groups hadnot obvious changed in intraperitoneal injection group and intranasaladministration group.4Compare the efficacy of PTX effects on dopaminergic systerm byintraperitoneal injection and intranasal administrationThe changes of the expressions of THmRNA、DATmRNA、TH and DATwere observed by RT-qPCR and immunoblotting; the density of DA and itsmetabolites HVA and DOPAC were analyzed by LC-MS. Intranansalpretreatment of PTX can bring every observation index recover to normallevel; intraperitoneal pretreatment of PTX only increased the contents ofDOPAV and HVA but did not recover to nomal levels, and the other indexesrecovered to normal level.Conclusion:1PTX enhanced the functions of SN-CPu DA neurons of rats.2The functions of SN-CPu DA neurons were weakened in Li-Pc induced ESrats.3The ameliorated effect of intranansal administration of PTX on SN-CPuDA neurons in Li-Pc induced ES rats is better than that of intraperitonealinjection.