Transforming Growth Factor Beta-1Reduces Spontaneous Recurrent Seizures And Inhibits Reactivation Of Glial Cells In Rats

Objective: Studies have demonstrated the neuroprotective activity of transforminggrowth factor beta-1(TGF-β1)，protecting neurons against different kinds of insults.However，the role of exogenous TGF-β1in the spontaneous recurrent seizures (SRS)、glial proliferation、 neuronal loss、 and mossy fiber sprouting followingpilocarpine-induced status epilepticus (SE) is unknown. The present study aimed todetermine the effect of intranasal TGF-β1administration，that is a novel, non-invasivedrug delivery strategy to central nervous system in which the BBB is bypassed, on SRSand explore the possible anti-epileptogenesis mechanisms.Methods: Male SD rats were divided into saline control group（Control group, n=25）,Pilocarpine model group(Pilo-group, n=25) and Pilocarpine model group pretreatedwith TGF-β1(TGF-β1group, n=25). Recombinant human TGF-β1was intranasallyadministered， Meanwhile, rats in the control group and the pilocarpine model groupwere treated identically with the same volume of PBS. Rats were injectedsubcutaneously with scopolamine to reduce the peripheral adverse effects of pilocarpine24h later after intranasally administered. Then, status epilepticus(SE) was triggered byintraperitoneal administration of pilocarpine30min later, chloral hydrate(10%,350mg/kg, i.p.) was administered50min following induction of SE to reducemortality rate. One week after SE, rats (each group, n=5) are put into a special cage to video monitored the average frequency, severity, and duration of SRSs12h per day for3weeks. Then these rats were killed to assessed aberrant mossy fiber sprouting (MFS),respectively. And other animals were killed at different time intervals after SE toquantified reactive astrocytosis using glial fibrillary acidic protein (GFAP) staining,microglia using Ionized calcium binding adaptor molecule1(Iba1) staining and neuronloss using Nissl staining in hippocampus, respectively.Results:1. TGF-β1reduces spontaneous recurrent seizures: No seizures were observedin the rats of Control group; We observed that the earliest spontaneous recurrentseizures were exhibited about9d in pilocarpine model group; While the earliestspontaneous recurrent seizures were exhibited about12d in TGF-β1group; Meanwhilethe average frequency, severity, and duration of SRSs were significantly reducedcompared with the pilocarpine model group.2. TGF-β1decreases Hippocampal GFAP and Iba1protein over-expression inPilocarpine-Induced Epilepsy. In the control group, there is a base glial expressed ofastrocytes showed slim morphology as well as thin and long processes, of microgliasshowed numerous and thin processes characteristic of the resting state of microglia weredistributed homogeneously in the hippocampus; In contrast, the density ofGFAP-positive and Iba1-positive cells in the entire hippocampus was significantlyincreased in the pilocarpine group animals, compared with Control controls. TheseGFAP-positive termed reactive gliosis, comprises altered morphology with hypertrophicsoma and processes, spatial overlapping and different functional changes of astrocytes,and Iba1-positive termed reactive microglia, comprises altered morphology with largeand round cell bodies with short and thick processes, which are characteristic features ofactive microglia and suggest phagocytosis activities. While in TGF-β1groups, these high density of reactive gliosis was significant decreased compared to pilocarpinegroups, which peaks at14d after SE，almost went to the baseline levels.3. TGF-β1inhibited the neuronal death： To further determine whether TGF-β1administered is sufficient to prevent pilocarpine-induced cell death, brains wereanalyzed to observe the surviving neurons using Nissl staining. The number of neuronsin the hippocampal CA3region was counted and compared with other groups. Ourresults showed that there was almost no apparent neuronal cell death in the Controlgroup, pilo-group reveals SE induced dramatic degeneration of neurons in thehippocampus, peaks at14d after SE, while the pretreatment of TGF-β1significantlyinhibited the neuronal death compared to Pilocarpine only treated group（p<0.05）.4. TGF-β1reduced mossy fiber sprouting in pilocarpine-induced Epileptic Rat. Timm’sstaining was used to determine whether TGF-β1could reverse mossy fiber sprouting inepileptic rats. The results indicated that little sprouting was present in the hippocampalCA3molecular layer in the control group，However, Timm’s staining showed robustmossy fiber sprouting in rats having a pilocarpine injection. This sprouting phenomenonwas reduced in rats with TGF-β1administration，that was less intense and moredispersed, though it is still more dense than that in the normal brain. These phenomenonsuggest that TGF-β1-treatment restrained pilocarpine-induced abberant MFS.Conclusions: Our data showed that intranasal administration of TGF-β1lead to reducethe average frequency, severity, and duration of spontaneous recurrent seizures，reduceover-expression of reactive gliosis, attenuate neuronal loss and inhibite abberant MFS inthe brain after pilocarpine-induced SE, which suggested a long-term protective role ofTGF-β1in the epileptic brain. The anti-epileptic and neuro-protective properties of TGF-β1may be related to its attenuating effect on over-expression of reactive gliosis.Moreover, we confirm that this non-invasive method of bypassing the BBB could bepreferentially delivered drugs to the brain, reducing unwanted systemic effects, andprovides a simpler, safer, potentially more cost-effective method of delivery than otherconventional methods currently in use.