Effects Of Intranasal TGF-β1on Contents Of Cytokines In The Hippocampus Of Rats With Status Epilepticus Induced By Pilocarpine

ObjectiveOur previous study has shown that exogeneous transforming growth factor beta1(TGF-β1) has a significant antiepileptic effect after status epilepticus (SE) is induced. Inthis study, a noninvasive intranasal administration was employed to deliver TGF-β1tothe central nervous system (CNS) of rats after pilocarpine-induced SE. Theconcentrations of pro-inflammatory cytokines including interleukin-1β (IL-1β), tumornecrotic factor (TNF-α), IL-6, and IL-18were measured in the hippocampus of rats afterSE was induced and the effects of TGF-β1on the hippocampal concentrations of thesepro-inflammatory cytokines were observed to explore the possible anti-inflammatorymechanism associated with the anti-epileptogenic function of TGF-β1.Material and MethodsExperiment1Detection of the concentration of TGF-β1in the brain afterintranasal administration of TGF-β1.8adult male SD rats were randomly divided into TGF-β1group and control group.There were four rats in each group. Rats in control group were given phosphate bufferedsaline (PBS), while rats in TGF-β1group were given TGF-β1. One hour after intranasaladministration, all of four rats in each group were sacrificed, the whole brain werecarefully excised, rinsed with cold PBS and snap frozen in liquid nitrogen. Theconcentration of TGF-β1in cerebral homogenate was measured by ELISA. T-test wasused for groups comparison, and P﹤0.05was considered have statistical significance. Experiment2Detection of the concentration of TGF-β1in the brain afterintranasal administration of TGF-β1.126adult male SD rats were included in the present study. SE was induced bypilocarpine in90rats. Diazepam (10mg/kg, i.p.) was administrated to terminate theseizure activity90min after the onset of SE. Three hours after pilocarpine inducedstatus epilepticus, rats were divided randomly into normal group, pilocarpine group(Pilo group) and pilocarpine+TGF-β1(TGF group). Then, rats of TGF group wereintranasally administered with TGF-β1, while rats of Pilo-group and Normal-groupgiven a same volume of PBS in the same way. Animals were executed and the brainswere harvested at24h,48h, and72h post-SE individually. Concentrations of IL-1β,TNF-α, IL-6, IL-18were detected in hippocampus via enzyme-linked immunosorbentassay (Elisa) and the immunohistochemisty (IHC). Then, the concentrations ofcytokines were analyzed with one-way analysis of variance (ANOVA) followed by theNewman–Keuls multiple comparison test. Probability values of﹤0.05wereconsidered significant.ResultsExperiment1Intranasal administration of TGF-β1elevated the concentration ofcerebral TGF-β1in rats.Compared with the control group, the concentration of TGF-β1in the brain wassignificantly increased1h after intranasal adminstration (P﹤0.01).Experiment2Intranasal administration of TGF-β1suppressed the hippocampalexpression of pro-inflammatory cytokines.We got the following results:①No positive cells of IL-1β, TNF-α，IL-6，IL-18wereobserved in Normal group.②24h after SE, IL-1β and TNF-α immunostaining was firstly expressed in cells with glia-like morphology in CA1of the hippocampus, whileIL-6and IL-18immunostaining was expressed in all regions of the hippocampus24hafter SE.③The immunoreactivity of IL-1β and IL-6was observed both in neurons andglia-like cells, while the immunoreactivity of TNF-α and IL-18was only observed inglia.④Compared with Pilo group, the mean optical density values of IL-1β, TNF-α,IL-6in the hippocampus were significantly reduced in TGF group72h after SE（P﹤0.05）. However, no difference in the expression of IL-18was found betweenTGF-group and Pilo-group at various time points after SE(P﹥0.05).⑤Comparedwith the normal control group, the contents of IL-1β、TNF-α、IL-6、IL-18weresignificant increased immediately after SE.⑥After intranasal administration ofTGF-β1, the contents of IL-1β, TNF-α, IL-6were significantly decreased at varioustime points (P﹤0.05or P﹤0.01), while no differences were found between Pilo-groupand TGF-group in that of IL-18at various time points after SE (P﹥0.05).⑦Concentrations of IL-1β、TNF-α、IL-6、IL-18in the hippocampus of pilocarpine-treatedrats were up-regulated after SE, and the change tendency between Pilo group and TGFgroup were similar.ConclusionBy the supplementation of intranasal TGF-β1after pilocarpine-induced SE, classicalpro-inflammatory cytokines (IL-1β, TNF-α, IL-6) were remarkably suppressed in thehippocampus. Thus we proposed that TGF-β1may exert its antiepileptogenic andneuropretective effects through regulating inflammatory cytokines after SE is induced.Intranasal administration is a noninvasive and practicable method to deliver TGF-β1tothe cental nervous systerm(CNS) bypassing blood-brain barrier, and this may offer apromising strategy for treating Epilepsy and probably other CNS disorders.