Cytokine Expression After Status Epilepticus In Immature Brain And Intervention Measures

BackgroundStatus epilepticus(SE) is defined as one continuous unremitting seizure lasting longer than 30 minutes, or recurrent seizures without regaining consciousness between seizures for greater than 30 minutes. It is one of the common emergencies in neurology department. The mortality rate and disability rate of SE is very high especially if treatment is not intiated quickly and properly. A high proportion of SE occur in younger people:37% occur in infants,73% in children younger than 3 years old,83% in children younger than 5 years old. Whether SE in childhood would change brain architecture and function and further influence intelligence and behavior development is a concern by parents and pediatricians. Previous clinical and animal studies indicated the immature brain is relatively resistant to seizure-induced brain damage. The mortality and incidence of sequelae following SE are low in children in the absence of an acute neurologic insult or progressive neurologic disorder. Similarly, long-term behavioral deficits or neuropathologic changes have not been found in immature rats in several seizure models. However, several two-hit seizure model studies demonstrate that the immature brain does not appear to be immune to injury following prolonged seizures. Schmid et al. reported that SE in adolescent rats with a history of neonatal seizures caused substantially more damage than in animals without a history of neonatal seizures. Likewise, Koh et al. found that although SE on postnatal day (P) 15 did not result in any detectable cell death or impairment of spatial learning, it predisposed rats to more extensive neuronal injury and worse performance in Morris water maze after the second seizure on P45. Recently, Somera-Molina explored the molecular events after kainic acid (KA)-induced early life seizures and found that transient microglial activation and persistent astrocyte activation may result in increased vulnerability to seizures in adulthood. However, the definite mechanism by which activated glia lead to increased vulnerability is unclear.Microglia and astrocyte activation are demonstrated as cell morphology change and expression of specific markers(Iba-1 for microglia and GFAP for astrocytes). Microglia activation is a complex process, including changes in pharmacological and electrophysiological properties, changes in migration and proliferation. Activated astrocytes also show many functional alterations, including glutamate release function, metabolism of glutamate, glutamate transport, water and potassium balance regulation. Apart from all above function changes, microglia and astrocytes are the two main sources of inflammatory factors in brain. More and more studies reveal that inflammation is involved in many acute and chronic brain diseases, including the diseases related to seizure. The expression of inflamamtory factors are elevated in resected tissue from temporal lope epilepsy patients. Likewise, inflammatory cytokines and markers for innate immunity(IL-1β, IL-6, TNFα, NF-κB system, COX-2, prostaglandins, Toll-like receptors) are upregulated in 1 hour after SE and persist for several days in hippocampus in temporal lope epilepsy animal models. The information about cytokine expression after seizure are mostly from studies in human adults and adult rats. There are few studies on immature brain. The cytokine expression after SE in immature brain have not been widely explored. The relationship between cytokine expression and increased vulnerability deserves further studies.Antiepileptic drugs are necessary after for children after SE in clinical practice. In this study, we will deliver several different antiepileptic drugs after SE in immature rats and observe whether these drugs would change cytokine expression and alleviate the increased vulnerability to second SE.Naloxone is a traditional drug in pediatrics, mainly used in neonatal resuscitation and wake promoting. Recent studies find that naloxone possesses inflammation-inhibitory and neuron protective property. Because transient microglial activation and persistent astrocyte activation appear after SE in P15 rats, we suppose that naloxone could inhibit inflammatory reaction in hippocampus after P15 SE and further change the increased vulnerability to second SE hit in adulthood.Objectives1. To investigate cytokine transcription and expression at acute and chronic stage after P15 SE.2. To explore the relationship of upregulated cytokines and increased vulnerability after P15 SE by application of cytokine antibodies.3. To observe after P15 SE, the effects of phenobarbital, depakine, topiramate, lamotrigine, levetiracetam monotherapy on chronic cytokine expression and vulnerability to second SE hit.4. To test the influence of naloxone on glia activation, cytokine expression and increased vulnerability after P15 SE.Methods1. Cytokine expression after P15 SE:P15 rats were randomly assigned to two groups:SE group(n=60) and Control group(n=60). Rats in SE group and Control group were further randomly divided into 6 groups(n=10 in each group) and sacrificed at 6 different time points for cytokine detection in hippocampus. Rats in SE group were intraperitoneally injected with kainic acid(KA) 5mg/kg to induce SE. Rats in control group were intraperitoneally injected with the identical volume of saline. Ten rats from each groups were sacrificed at 12h,24h,3d, 10d,20d,30d after administration of KA or saline. Hippocampal protein and mRNA were extracted. The cytokine(IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, TNFα, IFNγ)transcription level were determined by real-time PCR and the cytokine protein expression were determined by enzyme linked immunoabsorbent assay(Elisa).2. The relationship between increased cytokines(IL-1β, IL-10, TNFα) and increased vulnerability:P15 rats were randomly divided into SE group(n=10), Antibody intervention group(n=40) and Control group(n=10). Antibody intervention group were further divided into 4 intervention groups(n=10 in each group). Rats in SE group were given KA(5mg/kg injected i.p.) to induce SE. Rats in control group were injected intraperitoneally with the same volume of saline. Rats in four Antibody intervention groups were intracerebroventricularly injected with IL-1βantibody at 12h after SE, IL-1βantibody at 20d after SE, IL-10 antibody at 20d after SE, TNFa antibody at 20d after KA-induced SE at P15, respectively. Rats from all groups were injected with KA 15mg/kg intraperitoneally to induce SE at P45. Rats were tested for spatial learning and memory by Morris water maze on P50 and sacrificed on P55 for apoptosis detection by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).3. The effect of antiepileptic monotherapy on cytokine expression at chronic stage and increased vulnerability after P15 SE. P15 rats were randomly divided into six groups:SE group, phenobarbital group, sodium valproate group, topiramate group, lamotrigine group, levetiracetam group(n=20 in each group). Rats in SE group were injected with KA (5mg/kg) intraperitoneally to induce SE. Rats in four antiepileptic monotherapy groups were dosed with phenobarbital(30mg/kg/day), sodium valproate(200mg/kg/day), topiramate(40mg/kg/day), lamotrigine(50mg/kg/day), levetiracetam(200mg/kg/day) respectively for 7 days after P15 SE. At P 45, ten rats from each group were tested for astrocyte activation and cytokine expression. The residual ten rats from each group were injected with KA(15mg/kg) to induce second SE at P45 and went on TUNEL examination on P55.4. The protective effect of naloxone after P15 SE. P15 rats were divided into three groups:SE group, naloxone group and control group. Rats in SE group were injected with KA 5mg/kg to induce SE at P15. Rats from naloxone group were implanted subcutaneously with naloxone minipump of different doses after P15 SE. Rats from control group were intraperitoneally injected with saline. Glia activation states were examined by western blotting of Iba-1, GFAP and S100β. De novo synthesis of cytokines were assayed by real-time PCR and Elisa. Rats from each group were intraperitoneally injected with KA(15mg/kg) to induce SE at P 45 and compared for spatial learning ability in Morris water maze and neuron injury in TUNEL assay.Results1. Cytokine expression after P 15 SE. P 15 SE caused certain changes of cytokines in hippocampus at different time points. IL-1βwas significantly upregulated at both early(P<0.001) and chronic stage(P<0.001) after P15 SE. IL-10 and TNFαwere enhanced at chronic stage(P<0.01) after P15 SE compared with control group. However, the expression of IL-2, IL-4, IL-6, IL-12, IFNγwere not significantly changed after P15 SE at all time points examined(P>0.05).2. The relationship between increased cytokines(IL-1β, IL-10 and TNFα) and increased vulnerability to second SE. Rats in SE group performed worse in Morris water maze than rats in control group(P<0.001) and exhibited more apoptotic cells in hippocampus than rats in control group(P<0.01). Intracerebroventricular administration of IL-1βat both 12h and 20d after P 15 SE could improve performance in Morris water maze(P<0.01) and reduce the apoptotic cells in hippocampus after second SE at P45(P<0.05). Antagonism of IL-10 at 20 days after P15 SE could deteriorate learning ability(P<0.05) and aggravate apoptosis after second SE at P45(P<0.05). Administration of TNFαantibody intracerebroventricularly at 20 days after P15 SE improved performance in Morris water maze(P<0.05) and reduced the number of apoptotic cells in hippocampus after second SE at P45(P<0.01).3. The effect of antiepileptic monotherapy on glial activation, cytokine expression increased vulnerability after P15 SE. Phenobarbital or lamotrigine monotherapy after PI5 SE did not change the glia activation states, cytokines(IL-1β, IL-10, TNFα) expression and hippocampal vulnerability to second SE(P>0.05). Although sodium valproate monotherapy had no influence on glia activation states(P>0.05), it reduced expression of IL-1β,IL-10 and TNFα(P<0.05) and mitigate apoptosis after second SE (P<0.01). Glia activation states and cytokine(IL-1β, IL-10, TNFα) expression were not different between topiramate group and SE group(P>0.05). The number of apoptotic cells was significantly reduced in topiramate group compared with SE group(P<0.05). Glia activation states and expression of cytokines(IL-1β, TNFα) were mitigated by levetiracetam monotherapy(P<0.01). Tunel-positive cells in hippocampus was reduced by levetiracetam treatment(P<0.01).4. The protective effect of naloxone after P15 SE. Subcutaneous minipump administration of naloxone could inhibit Iba-1, GFAP and S100βupregulation at acute stage(P<0.01) and reduce GFAP and and S100βexpression in hippocampus at chronic stage(P<0.001). IL-β, TNFαand IL-10 expression in hippocampus were reduced in naloxone-treated rats(P<0.01). Rats with naloxone treatment exhibited better performance in Morris water maze after P 45 SE(P<0.01). The apoptotic cells in hippocampus were greatly reduced after second SE in nalxone-treated rats(P<0.01).Conclusions1. P15 SE could induce IL-1βexpression at acute stage in hippocampus and induce IL-1β, TNF-αand IL-10 expression at chronic stage in hippocampus.2. IL-1βproduction at acute and chronic stages after P15 SE increased the hippocampal vulnerability to second SE at P45. Increased IL-10 at chornic stage is a protective cytokine in hippocampus, alleviating the vulnerability to second SE at P45. Upregulation of TNFαat chronic stage after P15 SE aggravates hippocampal vulnerability to second SE at P45.3. Phenobarbital and lamotrigine monotherapy has no influence on increased vulnerability after P15 SE. Sodium valproate could decrease hippocampal vulnerability by inhibiting cytokine expression. Topiramate mitigate vulnerability to second SE through unknown mechanisms. Levetiracetam could decrease the vulnerability to second SE by depressing astrocyte activation and inhibiting cytokine expression.4. Naloxone treatment could alleviate glia activation and cytokine expression after P15 SE. Naloxone could alleviate the increased vulnerability to P 45 SE after P 15 SE.