Intranasal Administration Of Pentoxifylline Has A Preventive Role In Epilepsy Seizures Induced By Lithium-Pilocarpine

Epilepsy is one of the most common neurobehavioural disorders，whichmanifests sudden and repeated convulsions. Both transient and persistentseizures can cause the brain neurons damage and loss. Patients with epilepsyoften suffer from motor deficits as well as learning and memory problems.Recent studies show that epileptic seizure has a close relationship withdopamine (DA). Some experiments showed that striatal DA level and itsmetabolite dihydroxyphenylacetic acid (DOPAC) decreased in pilocarpineinduced younger or adult epilepsy rats by high-performance liquidchromatography analysis. DA and its metabolite homovanillic acid (HVA)decreased in cerebrospinal fluid of epilepsy patients and the decreased HVAwas improved after they experienced antiepileptic therapy. At present, themechanism of epilepsy seizures is not known. Some studies suggested adefinite role for reactive oxygen species (ROS) in the pathophysiology ofseizures. Neuronal hyperactivity is associated with excessive generation offree radicals and ROS can rapidly affect the structure and function of neurons.Inhibition of the over-excited neurochemical pathways can decrease theneuronal excitability, reduce the neuron loss and change the state of epilepticseizure, which is probably one of the neurobiological mechanisms of epilepsytreatment.Pentoxifylline (PTX) is a nonspecific phosphodiesterase inhibitor, has astrong antioxidant effect. This drug was initially introduced for the treatmentof respiratory and peripheral circulatory diseases. Recent studies have shownthe neuroprotective effect of PTX. PTX was used for the treatment of nervoussystem diseases, such as ischemic brain injury, neurotrauma and dementia.PTX was found to improve learning acquisition and memory following hippocampal lesions in rats as well. Another study found that intraperitonealpretreatment of animals with PTX significantly decreased the severity ofseizures and prolonged the latency to epileptic seizures, suggesting theantiepileptic and neuroprotective effects of PTX.The immediate termination of the seizure and the reduced damage to thenervous system are needed after epileptic seizures. Intravenous andintraperitoneal injection can’t be implemented immediately, that may delay thetiming of treatment and threaten the human life. Recent studies suggestedintranasal administration of PTX could achieve the same drug therapyconcentration as intravenous injection. Intranasal administration of drugs isconvenient, which has been become one of the preferred methods in recentyear. However, it is still unclear whether intranasal administration of PTX hasantiepileptic effect.Therefore, the male Wistar rats were used as experimental animal modelsin present study. The influence of intraperitoneal and intranasal administrationof PTX upon the central nervous system was studied by analyzing thebehavior of rats, the neuronal damage and mossy fibre sprouting (MFS) inhippocampus, and the tyrosine hydroxylase (TH) and dopamine transporter(DAT) expression in midbrain dopaminergic neurons. It is hoped thatintranasal administration of PTX could be used in treatment of epilepsy.Objective: To study the effect of intraperitoneal and intranasaladministration of PTX on TH and DAT expression in epileptic rats induced byLi-Pc, it is hoped that intranasal administration of PTX could be used inadjutant for some CNS diseases such as epilepsy.Methods: Male Wistar rats (300g) were randomly divided into5groups: control group (Con, n=15), lithium chloride group (Li, n=15),epileptic seizure group (ES, n=21), intraperitoneal adiministion ofpentoxifylline group (ES+PTX.ip, n=21), intranasal adiministion ofpentoxifylline group (ES+PTX.in, n=21). The animals in Con and Li groupreceived saline, Li (127mg/kg) alone, respectively. ES was induced in otherthree groups by administering an aqueous (saline) solution of lithium chloride (127mg/kg) intraperitoneally, followed (20hours later) by pilocarpine(15mg/kg) intraperitoneall. ES+PTX.ip group and ES+PTX.in group wasadministerstered PTX（60mg/kg） intraperitoneally and intranasal in30minbefore pilocarpine injection.The epileptic seizure of rats after the drug administration was observedby Racine score method. The neuronal damage and MFS in hippocampus wasanalyzed by Thionin and Timm staining, TH and DAT expressions weredetected by immunocytochemistry or immunoblotting.Results:1General observationThe Con and Li groups didn’t appear abnormal activity. After Pcadministration, The ES group of95.24%animals appeared seizure, with amean latency of17.18±5.20min to development of seizures; the ES+PTX.ipgroup of66.67%animals appeared seizures, with a mean latency of30.74±9.65min to development of seizures; the ES+PTX.in group of66.67%animals appeared seizures, with a mean latency of27.37±6.92min todevelopment of seizures. Our results showed that intraperitoneal andintranasal administration of animals with PTX significantly prolonged thelatency to seizures, increased by78%(P<0.01) and59%(P<0.01)respectively.2Thionin and Timm staining1) Thionin stainingNissl body is often called the chromophil substance, its main componentare ribonucleic acid (RNA) and protein, which can be stained purple blue bythionine. The nerve cells in the CA1, CA3and hilus areas was observed underlight microscope after thionine staining. The neurons in Con and Li rats hadcomplete, clear boundary and rich in Nissl body. The cells in the ES grouppartly lost, arranged in disorder and Nissl body decreased in the CA1,CA3and hilus areas of hippocampus compared with Con group, also the pyramidaland granule cell layer of the dentate gyrus neurons reduced(P<0.01).Compared with Con group, the intraperitoneal and intranasal administration of PTX had a attenuating effect, preventing cell loss in all threeregions(P<0.05).2) Timm stainingThe mossy fiber terminals of dentate granule cell is rich in Zn2+, whichcan be stained by silver sulfide, appears as a dark brown or black particles.Compared with the Con group, the rats in ES group had increased Timmgranules in the CA3(P<0.01), and had no significant differences in themolecular layer of dentate gyrus. Administration of PTX reduced the MFS inthe CA3(P<0.01).3Immunocytochemistry1) The influence of PTX on TH expression in ES ratsThe statistics analysis showed that, the expression of TH in ES ratsdecreased by19%(P<0.01) in SN,12%(P<0.01) in CPu and11%(P<0.05) inVTA compared with the Con group. The Li group, ES+PTX.ip group andES+PTX.in group showed no significant difference with the Con group. PTXdelivery to rats improved the reduced expressions of TH induced by epilepticseisure. Compared with ES group, the expression of TH in ES+PTX.ip ratsincreased by12%(P<0.01)in SN,15%(P<0.01) in CPu and11%(P<0.05) inVTA respescively; the expression of TH in ES+PTX.in rats increased by25%(P<0.01) in SN,14%(P<0.01) in CPu and12%(P<0.05) in VTA respescively.2) The influence of PTX on DAT expression in ES ratsThe statistics analysis showed that, the expression of DAT in ES ratsdecreased by21%(P<0.01)in SN,6%(P<0.05) in CPu and26%(P<0.01) inVTA compared with the Con group. The Li rats showed no significantdifference with the Con rats. Compared with the ES group,the expression ofDAT in ES+PTX.ip rats increased by51%(P<0.01)in SN,16%(P<0.01) inCPu and34%(P<0.01) in VTA respescively; the expression of DAT inES+PTX.in rats increased by48%(P<0.01)in SN,17%(P<0.01) in CPu and28%(P<0.01) in VTA respescively.4Immunoblotting:1) The influence of PTX on TH expression in ES rats The statistics analysis showed that, the expression of TH in ES rats decr-eased by32%(P<0.01)in SN,21%(P<0.01) in CPu and24%(P<0.01) inVTA compared with the Con group respectively. The Li rats showed nosignificant difference with the Con rats. The expression of TH in ES+PTX.iprats increased by68%(P<0.01) in SN,41%(P<0.01) in CPu and51%(P<0.05) in VTA compared with ES group, and higher than Con rats in SNand VTA. The expression of TH in ES+PTX.in rats increased by58%(P<0.01)in SN,48%(P<0.01) in CPu and56%(P<0.05) in VTA compared with ESrats and higher than Con rats in VTA.2) The influence of PTX on DAT expression in ES ratsThe statistics analysis showed that, the expression of DAT in ES ratsdecreased by20%(P<0.01) in SN,15%(P<0.01) in CPu and15%(P<0.01) inVTA compared with the Con group respectively. The Li rats showed nosignificant difference with the Con rats. The expression of DAT in ES+PTX.iprats increased by42%(P<0.01) in SN,40%(P<0.01) in CPu and47%(P<0.05) in VTA compared with ES group, and higher than Con rats in thethree brain regions. The expression of DAT in ES+PTX.in rats increased by43%(P<0.01)in SN,37%(P<0.01) in CPu and43%(P<0.05) in VTAcompared with ES rats and higher than Con rats in the three brain regions..Conclusion:1PTX decreased the rate of seizures, prolonged the latency to epilepticseizures, and reduced the neuronal damage and MFS in hippocampus,suggesting the antiepileptic and neuroprotective effects of PTX.2The expression of TH and DAT was decreased in Li-Pc induced ES ratsand the PTX delivery to rats improved the reduced expressions of TH andDAT induced by epileptic seisure.3Intranasal administration of PTX has the preventive effect on the epilepticseizure as the intraperitoneal injection, suggesting it can be another route toprevent the epilepsy.