| Epilepsy, resulted from the super-excitability and the synchronous firing of neurons, is a serious chronic disease which will result in the transient abnormality of the central nerve system, which mainly manifests sudden and repeated convulsions. Status epilepticus(SE) is an emergency neurological condition where recurrent generalized seizures last for more than 30 minutes and if not controlled neuronal injury occurs. Patients or animal models with epilepsy often suffer from congnitive deficits, and mainly manifest learning and memory impairment. Morever, The nigrostriatal and mesocorticolimbic DAergic pathway have been shown to play a critical role in memory and cognition. In addition to the traditionally glutamatergic and inhibitory neurotransmitter, studies have shown that the dopaminergic system is also involved in seizure onset and spread. Such as a decreased dopamine(DA)content in hippocampus and striatum and the reduced dopamine transporter(DAT) in substantia nigra(SN) and putamen, etc. Therefore, the protection of dopaminergic systems has been suggested as a therapeutic strategy for the SE-induced neurobehavioral deficits.The mechanism of epilepsy is very complex. The oxidative stress induced oxygen free radical(FR) chain reaction is the core of the seizures and neurodegenerative changes. Oxidative stress is due to the imbalance between oxidation and antioxidation in vivo, which leads to excessive free radicals and(or) decreased scavenging capacity. In the central nervous system(CNS), free radicals are mainly produced by the metabolism of neurotransmitters and excitatory amino acids. Neuronal hyperactivity and/or excitotoxicity have been associated with excessive generation of free radicals, reactive oxygen species(ROS) can dramatically affect the structure and function of neurons.Meanwhile, oxidative stress is one of the mechanisms implicated in the progression of dopaminergic degeneration. Study has found the amelioration effect on age-related DAergic function decline through activating the antioxidant system in SN and ventral tegmental area(VTA). Therefore, in view of the participation of the oxygen free radicals in the epileptic, improvement of the antioxidant capacity may be one strategy for the declined DAergic function induced by epilepsy.The nuclear factor erythroid 2-related factor 2(Nrf2) is the key factor in cell oxidative stress reaction, recent studies have found that Nrf2 mediates and activates a variety of phase II detoxification enzymes and antioxidant gene transcription through Keap1-Nrf2-ARE signaling pathway, thereby reducing oxidative stress induced cell damage, maintaining the homeostasis of the oxidation and antioxidant system in the body. Nrf2 is located in the cytoplasm combined with Keap1 in the physiological state, when the body was attacked by such as oxygen free radicals, the Nrf2 dissociated with Keapl, and the half-life of Nrf2 extended obviously, then Nrf2 transposition into the nucleus,and combined with the ARE inducing the expression of a group of cytoprotective enzymes, for example, Heme oxygenase 1(HO-1) and NADPH:quinone oxidoreductase1(NQO1), enhance the antioxidant capacity of cell and protect cells from the toxic injury. Activation of this pathway increases total protein expression and nuclear levels of Nrf2. Disruption of Nrf2 signaling is associated with an increased susceptibility to oxidative insults and other toxicants in humans and model organisms. Previous studies have shown that up-regulation of HO-1 or NQO1 in neurons can protect them against oxidative and excitotoxic insults. Recent studies demonstrated that the activation of Nrf2-ARE signal pathway with sulforaphane(SF) in hippocampus can suppress the progression of chronic amygdala kindling, and ameliorate the cognitive impairment and oxidative stress induced by epileptic seizure.Pentoxifylline(PTX) is an adenosine antagonist and nonspecific type of4-phosphodiesterase inhibitor. It is a potent antioxidant and modulator of avariety of transmitters and penetrates the blood–brain barrier rapidly and efficiently after systemic administration. This drug was initially introduced for the treatment of peripheral circulatory and respiratory disorders, primarily because of its hemorrheologic effects. Recent studies have shown the neuroprotective effect of PTX against a host of neurobehavioral disorders induced by ischemic brain injury and and stroke. PTX was found to improve learning and and memory in glutamate-lesioned rats, intraperitoneal injection with PTX has ameliorated effects on the seizure activities and hippocampal neural injury of SE rats induced by Li-Pc, but its mechanism is not yet clear.The character of epilepsy is sudden attack that warrants immediate treatment to terminate the seizures activity especially the SE and prevent associated neuronal damage. Oral, intravenous and intraperitoneal injection can’t be absorbed or implemented immediately, this may delay or miss the best treatment time and threaten the human life. Previous studies demonstrated that intranasal midazolam, lorazepam and diazepam are effective at terminating prolonged seizures, preventing seizure clustering and avoiding post-ictal behavioural complications in adolescent and adult patients with severe epilepsy, which appear to be a safe and effective rescue treatment for pilepsy patients. To date, it is not known whether intranasal PTX administration would be effective to the epileptic rats.The epilepsy rat model induced by Li-Pc showed similar pathological and clinical features of humanity temporal lobe epilepsy(TLE), it is a ideal model of TLE and suitable for exploring the mechanism of epilepsy and screening antiepileptic drugs(AEDs). Therefore, epileptic rats induced by Li-Pc were used in the present study to examine the effects of intranasal PTX supplement on seizures and cognitive impairments; evalue the dopaminergic function in ventral midbrain which which has relative relation with cognition; detect the oxidative stress parameters, Nrf2, HO-1 and NQO1 in related brain region;examine the c AMP levels in the brain region mentioned above. It is hoped that the results provide experimental evidence for the pathogenesis of epilepsy, and provide a new approach for the treatment of epilepsy with PTX.Part1 PTX reduces Li-Pc induced seizures and improves cognitive dysfunction in ratsObjective: To study the effects of intranasal and intraperitoneal administration PTX on the seizures and cognitive impairment in rats induced by lithium-pilocarpine.Methods: Epileptic rats induced by Li-Pc were used in the present study,pretreatment with PTX by intranasal administration(In PTX) or intraperitoneal injection(Ip PTX); evaluate the epileptic seizures in rats by statisticsing the latency to seisures, the latency to SE and so on; explore the cognitive impairment in rats through using the Morris test; investigate the MFS in hippocampal CA3 of rats by Timm staining; detected the content of DA and its metabolites in hippocampus of rats by HPLC.Results:1 There was no abnormality in rats of Con-in group, Li Cl-in group,PTX-in group, Con-ip group, Li Cl-ip group, and PTX-ip group. Within 6 or 5minutes after the injection of Pc, animals started developing a gradual and significant change in behavior including PCS(miosis, piloerection, diarrohea,mild tremors, scratching, salivation) and STM(sniffing, paw licking and rearing), followed by seizures in 100% of animals in SE-in group and SE-ip group, with a mean latency of 11.92 ± 3.38 minutes and 10.77 ± 3.52 minutes to develop seizures respectively; followed by SE in 85.71% and 80.95% of animals in SE-in group and SE-ip group respectively, with a mean latency of24.07 ± 2.17 minutes and 25.19 ±4.41 minutes to develop SE respectively. The mortality was observed over a period of 24 h following Pc injections, in ES-in group and ES-ip group, 9.52% of the rats died within 24 hours.PTX pretreatment reduced the frequency and intensity of PCS, STM and seizure episodes. and PTX pretreatment increased the latency to seizures and SE and decreased the percentage of seizures and SE significantly. Compared to the SE-in group and SE-ip group, the latency to seizures of group PTX+SE-in(23.81%) and group PTX+SE-in(38.10%)were significantly increased(P<0.01). Furthermore, no rat develop SE and no mortality wasobserved in the rats pretreated with PTX, as compared with 9.52% mortality in the SE-in group and SE-ip.2 Morris water mazeIntranasal administration group: All groups displayed gradual improvement in the escape latency over the 5-day testing period. ES-in rats exhibited longer escape latencies to reach the platform as compared with the Con-in rats(P<0.05 on 1st day and 2nd day, P<0.01 on 3rd to 5th day;however, the poor performance was mitigated by intranasal PTX treatment(P<0.05 on 2nd day, P<0.01 on 1st day and 3rd to 5th day). In the probe trial,SE-in rats spent significantly less the time in the target quadrant than the Con-in rats(P<0.01), while intranasal PTX treatment significantly improved the performance(P<0.01). The number of crossing the platform in the SE-in rats obviously decreased as compared to the Con-in rats(P<0.01), while intranasal PTX treatment markedly increased the number of crossing(P<0.01).In addition, there was no significant effect on cognition among Con-in,Li Cl-in and PTX-in rats.Intraperitoneal administration group: The effects of PTX intraperitoneal injection on cognition was consistent with intranasal PTX treatment.3 Timm stainingIntranasal administration group: SE-in rats showed a modest sprouting of mossy fibers in the CA3 area as compared with Con-in rats, while intranasal PTX treatment attenuated the sprouting of the mossy fibers. Group difference was shown among the Con-in, Li Cl-in, PTX-in, SE-in and PTX+SE-in rats in Timm score of CA3 area(χ2=15.579, P<0.01). The post hoc test showed that the Timm score was significantly increased in SE-in rats compared to Con-in rats(P<0.01). The Timm score was significantly decreased in PTX+SE-in rats compared to SE-in rats(P<0.01). There was no significant effect on the Timm score among Con-in, Li Cl-in and PTX-in rats(P>0.05).Intraperitoneal administration group: Compared to the SE-ip group,intraperitoneal injection with PTX significantly increased the Timm score(P<0.01).4 DA and its metabolitesIntranasal administration group: Group differences were shown among the Con-in, Li Cl-in, PTX-in, SE-in and PTX+SE-in rats in Hip(DA:F(4,22)=61.733, P<0.01; DOPAC: F(4,22)=92.890, P<0.01; HVA:F(4,22)=40.507, P<0.01) on DA, DOPAC and HVA. The post hoc test indicated that DA, DOPAC and HVA were significantly reduced in Hip of SE-in rats compared to Con-in rats(P<0.01) and increased by 93%(P<0.01),216%(P<0.01) and 51%(P<0.01) in Hip of PTX+SE-in rats compared to the SE-in rats respectively. DA and its metabolites above in PTX+SE-in rats reached the level of SE-in rats. There were no significant differences in DA,DOPAC and HVA of Hip among the Con-in, Li Cl-in and PTX-in rats.Intraperitoneal administration group: DA, DOPAC and HVA of PTX+SE-ip rats significantly increased by 86%(P<0.01), 160%(P<0.01) and53%(P<0.01) respectively in Hip compared to the SE-ip rats.Summary:1 The epilepsy rats induced by lithium-pilocarpine accompanied with cognitive impairment.2 Pretreatment of PTX reduced the seizures and ameliorated the cognitive impairment in rats induced by lithium-pilocarpine.3 Pretreatment of PTX decreased the MFS and reduced the lesion of dopaminergic innervation in hippocampus of rats induced by lithium-pilocarpine.4 Pretreatment of PTX ameliorated the decreased DA and its metabolites in rats induced by lithium-pilocarpine.5 Nasal route can be used as a way to treat epilepsy.Part2 The amelioration effect of PTX on dopaminergic neurons in the midbrain of epilepsy rats induced by Li-PcObjective: To study the effect of PTX administration on dopaminergic activity in Ventral midbrain(VM), Caudate-putamen(CPu) and Hippcampus(Hip) of epilepsy rats induced by Li-Pc.Methods: The RT-PCR was used to detect the TH m RNA and DATm RNA in VM; the Western blot was used to detect the TH and DAT protein in VM and Hip; the HLPC-MS was used to analyse the effects of PTX administration on the level of DA and its metabolites in CPu of rats.Results:1 RT-PCRIntranasal administration group: Group differences in TH m RNA(F(4,22)=72.686, P<0.01) and DAT m RNA(χ2=17.715, P<0.01) were detected in VM among the Con-in, Li Cl-in, PTX-in, SE-in and PTX+SE-in rats. The post hoc test showed that TH m RNA and DAT m RNA were significantly lower in SE-in rats compared to Con-in rats(P<0.01). Both m RNAs were in the same level of PTX+SE-in rats as in Con-in rats. TH m RNA and DAT m RNA of PTX+SE-in rats significantly increased by 48%(P<0.01) and 50%(P<0.01) compared to SE-in rats. There was no significant effect on both m RNAs among Con-in, Li Cl-in and PTX-in rats.Intraperitoneal administration group: TH m RNA and DAT m RNA of PTX+SE-ip rats increased by 42%(P<0.01) and 47%(P<0.01) in SN respectively compared to SE-ip rats.2 Western BlotIntranasal administration group: Western blot was used to detect the protein level of TH and reduced DAT extracted from VM, CPu and Hip. TH was located at ~60k Da, while reduced DAT was located at ~80 k Da. There were group differences of TH and DAT levels found among the Con-in,Li Cl-in, PTX-in, SE-in and PTX+SE-in rats in VM(TH: F(4,22)=144.914,P<0.01; Fig. 3E, DAT: χ2=14.314, P<0.01), CPu(TH: F(4,22)=50.721,P<0.01; Fig. 3H, DAT: χ2=16.280, P<0.01) and Hip(TH: F(4,22)=18.219,P<0.01; Fig. 3K, DAT: χ2=17.015, P<0.01). The post hoc test revealed that TH and DAT in VM, CPu and Hip of SE-in rats were significantly reduced compared to Con-in rats(P<0.01). There was no difference of TH and DAT in VM, CPu and Hip between PTX+SE-in rats and Con-in rats. TH and DAT of PTX+SE-in rats significantly increased by 282%(P<0.01) and 173%(P<0.01)in VM, by 190%(P<0.01) and 100%(P<0.01) in CPu, as well as by 63%(P<0.01) and 290%(P<0.01) in Hip respectively compared to SE-in rats.There was no significant effect on TH and DAT in VM, CPu and Hip among the Con-in, Li Cl-in and PTX-in rats.Intraperitoneal administration group: TH and DAT of PTX+SE-ip rats increased by 270%(P<0.01) and 182%(P<0.01) in VM, by 144%(P<0.01)and 72%(P<0.01) in CPu, as well as by 40%(P<0.01) and 205%(P<0.01) in Hip respectively compared to SE-ip rats.3 HLPC-MSIntranasal administration group: Group differences were shown among the Con-in, Li Cl-in, PTX-in, SE-in and PTX+SE-in rats in CPu(DA:F(4,22)=49.223, P<0.0 DOPAC: F(4,22)=29.512, P<0.01; HVA:F(4,22)=33.023, P<0.01) on DA, DOPAC and HVA. The post hoc test indicated that DA, DOPAC and HVA were significantly reduced in CPu of SE-in rats compared to Con-in rats(P<0.01) and increased by 74%(P<0.01),33%(P<0.01) and 68%(P<0.01) in CPu of PTX+SE-in rats compared to the SE-in rats respectively. DA and its metabolites above in PTX+SE-in rats reached the level of SE-in rats. There were no significant differences in DA,DOPAC and HVA of CPu the Con-in, Li Cl-in and PTX-in rats.Intraperitoneal administration group: DA, DOPAC and HVA of PTX+SE-ip rats significantly increased by 67%(P<0.01), 43%(P<0.01) and86%(P<0.01) respectively in CPu compared to the SE-ip rats.Summary:1 The dopaminergic activity declined in midbrain of epilepsy rats induced by Li-Pc.2 PTX pretreatment ameliorated dopaminergic activity in midbrain of epilepsy rats induced by Li-Pc.3 The amelioration effect of intranasal supplement with PTX on the declined dopaminergic activity in midbrain of epilepsy rats induced by Li-Pc was superior to intraperitoneal injection.Part3 Pentoxifylline region-specifically activited Nrf2-ARE pathway in epileptic rat induced by lithium-pilocarpineObjective: The present study was aimed to evaluate if PTX could ameliorate oxidative stress via activating the Nrf2-ARE signal pathway and/or increasing the c AMP level, explore the he protective mechanism of PTX on the dopaminergic neurons in epilepsy rats induced by Li-Pc.Methods: Detect the oxidative stress parameters(MDA, LPO, GSH, SOD)in VM and Hip by the spectrophotometry; analyse the Nrf2 m RNA, HO-1m RNA and NQO1 m RNA in VM and Hip by RT-PCR; measure the Nrf2,HO-1 and NQO1 protein in VM and Hip by Western blot; determine the c AMP level in VM and Hip by radioimmunoassay.Results:1 Oxidative stress parametersIntranasal administration group: There were group differences found among the Con-in, Li Cl-in, PTX-in, SE-in and PTX+SE-in rats on the level of MDA(VM: χ2=17.033, P<0.01; Hip: F(4,22)=35.598, P<0.01), LPO(VM:χ2=16.419, P<0.01; Hip: χ2=16.693, P<0.01), GSH(VM: F(4,22)=66.059,P<0.01; Hip: F(4,22)=68.995, P<0.01) and SOD(VM: F(4,22)=37.864,P<0.01; Hip: F(4,22)=12.912, P<0.01) in VM and Hip. The post hoc test revealed the significantly increased MDA and LPO as well as reduced GSH in VM, CPu and Hip of SE-in rats compared to Con-in rats(P<0.01). MDA,LPO, GSH and SOD in VM and Hip of PTX+SE-in rats were in the same level as Con-in rats. MDA decreased by 14% and 23%(P<0.01), LPO decreased by 31% and 30%(P<0.01), GSH increased by 78%(P<0.01) and76%(P<0.01) as well as SOD increased by 25%(P<0.01), 23%(P<0.01) and19%(P<0.01) in VM and Hip of PTX+SE-in rats compared to that in SE-in rats respectively. There were no significant differences in MDA, LPO, GSH and SOD of VM and Hip among the Con-in, Li Cl-in and PTX-in rats.Intraperitoneal administration group: MDA decreased by 19% and 23%(P<0.01), LPO decreased by 24% and 28%(P<0.01), GSH increased by 101%and 86%(P<0.01) as well as SOD increased by 21% and 22%(P<0.01) in VM and Hip of PTX+SE-ip rats compared to that in SE-ip rats respectively.2 RT-PCR Part3 Pentoxifylline region-specifically activited Nrf2-ARE pathway inepileptic rat induced by lithium-pilocarpineIntranasal administration group: Group differences among the Con-in,Li Cl-in, PTX-in, SE-in and PTX+SE-in rats were found in Nrf2 m RNA(VM:F(4,22)=21.305, P<0.01; Hip: F(4,22)=65.829, P<0.01), HO-1 m RNA(VM:F(4,22)=21.598, P<0.01; Hip: F(4,22)=37.175, P<0.01) and NQO1 m RNA(VM: χ2=15.963, P<0.01; Hip: F(4,22)=66.175, P<0.01) in VM and Hip. The post hoc test showed that Nrf2 m RNA, HO-1 m RNA and NQO1 m RNA in VM and Hip were significantly reduced in SE-in rats compared to Con-in rats(P<0.01). There were no significant differences of Nrf2 m RNA, HO-1 m RNA and NQO1 m RNA in VM between PTX+SE-in rats and Con-in rats rats. Nrf2 m RNA, HO-1 m RNA and NQO1 m RNA significantly increased by 60%(P<0.01), 38%(P<0.01) and 90%(P<0.01) in VM of PTX+SE-in rats compared to SE-in rats respectively. There were no significant differences of Nrf2 m RNA, HO-1 m RNA and NQO1 m RNA in Hip between SE-in rats and Con-in rats rats. In addition, there were no significant differences in Nrf2 m RNA, HO-1 m RNA and NQO1 m RNA of VM and Hip among the Con-in,Li Cl-in and PTX-in rats.Intraperitoneal administration group:Nrf2 m RNA, HO-1 m RNA and NQO1 m RNA significantly increased by 50%(P<0.01), 41%(P<0.01) and82%(P<0.01) in VM of PTX+SE-ip rats compared to SE-ip rats respectively.3 Western blotIntranasal administration group: Group differences of the Nrf2, HO-1 and NQO1 among the Con-in, Li Cl-in, PTX-in, SE-in and PTX+SE-in were found in VM(Nrf2: F(4,22)=84.169, P<0.01; HO-1: F(4,22)=71.535, P<0.01; Fig.5G, NQO1: χ2=17.290, P<0.01) and Hip(Nrf2: F(4,22)=65.157, P<0.01;HO-1: F(4,22)=82.190, P<0.01; NQO1: F(4,22)=67.932, P<0.01). The post hoc test showed that Nrf2, HO-1 and NQO1 in VM and Hip were significantly reduced in SE-in rats compared to Con-in rats(P<0.01). There were no significant differences of Nrf2, HO-1 and NQO1 in VM between PTX+SE-in rats and Con-in rats, except for NQO1 in VM that were lower in PTX+SE-in rats than Con-in rats(P<0.05). Nrf2, HO-1 and NQO1 significantly increased by 262%(P<0.01), 121%(P<0.01) and 65%(P<0.01) in VM of PTX+SE-inrats compared to SE-in rats respectively. There were no significant differences of Nrf2, HO-1 and NQO1 in Hip between SE-in rats and Con-in rats rats. In addition, there were no significant differences in Nrf2, HO-1 and NQO1 of VM and Hip among the Con-in, Li Cl-in and PTX-in rats.Intraperitoneal administration group: Nrf2, HO-1 and NQO1 significantly increased by 252%(P<0.01), 129%(P<0.01) and 36%(P<0.01) in VM of PTX+SE-ip rats compared to SE-ip rats respectively.4 c AMPThere were group differences found among the Con-in, Li Cl-in, PTX-in,SE-in and PTX+SE-in rats on the level of c AMP in VM(F(4,22)=9.829,P<0.01) and Hip(χ2=15.319, P<0.01). The post hoc test revealed the significantly reduced c AMP in VM and Hip of SE-in rats compared to Con-in rats(P<0.01). c AMP in VM and Hip of PTX+SE-in rats were in the same level as Con-in rats. c AMP increased by 32%(P<0.01), 11%(P<0.01) and26%(P<0.01) in VM and Hip of PTX+SE-in rats compared to that in ES-in rats respectively. There were no significant differences in c AMP of VM, CPu and Hip among the Con-in, Li Cl-in and PTX-in rats.Intraperitoneal administration group: c AMP in VM and Hip of PTX+SE-ip rats were in the same level as Con-in rats. c AMP increased by32% and 26%(P<0.01) in VM, CPu and Hip of ES+PTX-ip rats compared to that in ES-ip rats respectively.Summary:1 Increased oxidative damage and decreased antioxidation capacity were found in brain of epilepsy rats induced by Li-Pc.2 PTX pretreatment specifically activated the Nrf2-ARE signal pathway in VM and ameliorated the oxidative stress state in brain of epilepsy rats induced by Li-Pc.3 PTX pretreatment improved the c AMP level in VM and Hip in brain of epilepsy rats induced by Li-Pc.4 The amelioration effect of intranasal supplement with PTX on the oxidative stress of epilepsy rats induced by Li-Pc was superior tointraperitoneal injection.Conclusions:1 Pretreatment of PTX reduced the seizures and ameliorated the cognitive impairment and dopaminergic activity in midbrain of rats induced by lithium-pilocarpine.2 Pretreatment of PTX specifically activated the Nrf2-ARE signal pathway and ameliorated the oxidative stress state in brain of epilepsy rats.3 The amelioration effect of intranasal supplement with PTX on the seizures of rats induced by Li-Pc was superior to intraperitoneal injection. |
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