| Background and objective:Epilepsy is one of the most common neurological disorders affecting children. Mesial temporal lobe epilepsy (MTLE) is the most common epilepsy subtype and now considered as an important example for the medically intractable epilepsies. Hippocampal sclerosis is a frequent pathological hallmark of MTLE. The initial method to treat MTLE is using antiepileptic drugs to reduce epileptic seizures aiming to decrease the brain damage caused by recurrent convulsions. Surgical resection of the affected area is now an important modality in treatment of drug resistant MTLE. Even a percentage of patients treated with selective amygdalohippocampectomy will continue to suffer from recurrent seizures. Because lacking of in-deep understanding to the molecular MTLE pathogenesis, there is no available curative medical treatment.Epilepsy in children is quite different from epilepsy in adults. Seizure susceptibility reaches the peak during the intense period of rapid brain growth and synaptogenesis that occurs in the immature brain. Although epilepsy may become manifest at any age, the onset of its causative lesion often occur in the first years of life, when the nervous system is still immature and highly susceptible to abnormal metabolic, traumatic and inherited damaging factors. The phases of neural maturation during which such factors exert their influence seem to be a key factor in determining the threshold for the development of any kind of epileptic seizure, either convulsive or non-convulsive. In the last20years, researchers found that immune system activation, transition of inflammation play important and persistent roles in the process of chronic spontaneous seizures development in epilepsy. It is also reported that in the developing brain, inflammatory brain response associated with the nervous system would increase the frequency seizures. Therefore, study the causal relationship between brain inflammation and epilepsy development represent an important step in understanding the molecular pathophysiology of MTLE development.Interleukin-1β (IL-1β), is a highly expressed pro-inflammatory cytokine that expressed in the hippocampus of epileptic patients and epilepsy animal models, which also suspected to play an important role in the epileptogenic process. IL-1β can be induced by reactive astrocytes, which in turn trigger downstream cascade of inflammatory reaction. This inflammatory reaction plays a vital role in transformation of normal brain to epileptic one with chronic recurrent spontaneous seizures. What factors leads to the astrocytes activation and IL-1β secretion, and how IL-1β works within the central nervous system (CNS) and promote the progress of chronic MTLE? Till now it is not clear. This study tried to explore the causal relationship between inflammation and pathogenesis of MTLE, and attempts to search for its role in the development of chronic epileptic condition. Hoping to open new avenue in understanding pathogenesis of intractable MTLE which may leads to improvement in this disease management and prognosis.Methods:The research includes three parts:1. Postnatal25day Sprague-Dawley (SD) rats were subjected to intraperitoneal injection (i.p) of pilocarpine to induce status epilepticus (SE) which progress to develop MTLE rat model. To validate the occurrence of recurrent spontaneous seizures and development of chronic stage of MTLE, electroencephalogram (EEG) was recorded to observe the epileptic discharges, Nissl and Timm staining were performed to evaluate neurons loss and mossy fibre sprouting (MFS). Cell models of high purity astrocyte and neuron cultures were used to perform the subsequent in vitro experiments.2. Western blot (WB), Real time quantitative RT-PCR (qPCR), Electrophoretic Mobility Shift Assay (EMSA) and Enzyme Linked Immunosorbent Assay (ELISA), Immunological Histological Chemistry (IHC), fluorescent double labelling were used to test the expression of myeloid-related protein8(MRP8), Toll-like receptor4(TLR4), nuclear factor kappa B (NF-kB), and IL-1β in hippocampal tissues obtained from MTLE patients and rat model. MRP8, Pyrrolidinedithiocarbamic acid (PDTC) and lipofection transfection were used stimulate and inhibit TLR4in pre-treated astrocyte, then the expressions of TLR4, NF-κB and IL-1β were detected and the direct relationship between these cytokines were analyzed.3. Using WB, co-immunoprecipitation (co-IP) and FM4-64dye to test the expressions of PI3K-p85, p-Akt, p-p70S6k and PI3K-p85combined with Interleukin-1receptor (IL-1RI) in hippocampal tissues of MTLE patients and rat model. IL-1β, Rapamycin and lentiviral transfection to inhibit PI3K-p85were used to pre-treat neurons, then the expressions of PI3k-p85、p-Akt、p-p70S6k and MAP2were detected and the relationship among the tested cytokines were analyzed. Neurons endocytosis were observed in each group.Results:1. Status epilepticus (SE) and epileptic seizures of MTLE rat model could be induced by intraperitoneal injection of pilocarpin. And after a period of seizure-free latent period, chronic onset of spontaneous was observed in MTLE rat model. The EEG shown typical epileptiform discharge; a widespread cell loss, degeneration, reactive gliosis and plasticity of neurons were noted in the hippocampus by nissl staining (p<0.05, vs control group), and Timm staining found abnormal mossy fiber terminal staining increased in inner molecular layer of dentate gyrus (p<0.05, vs control group). High purity of AS and neuron were also successfully cultured.2. The expressions of MRP8, TLR4, NF-κB and IL-1β were increased in the hippocampus of MTLE patients (p<0.05, vs control group), they also up-regulated in the acute and chronic stage but not latent stage of rat model (p<0.05, vs control group). MRP8increased TLR4, NF-κB and IL-ip expressions in astrocyte (p<0.05, vs control group) and NF-κB transferred into the nucleus which could inhibited by PDTC and silence of TLR4. On the other side, increase of TLR4expression induced by MRP8was not affected by PDTC (p<0.05, vs MRP8group).3. The expressions of PI3K-p85, p-Akt, p-P70S6K and PI3K-p85combined with IL-1RI were increased in the hippocampus of MTLE patients (p<0.05, vs control group), and in the three stages of MTLE rat model (p<0.05, vs control group). IL-1β increased PI3K-p85, p-Akt, p-p70S6k and PI3K-p85combined with IL-1RI expressions in neuron and increase the neurons endocytosis (p<0.05, vs control group) which could inhibited by Rapamycin and silence of PI3K-p85(p<0.05, vs IL-1β) group. To inhibit the combining PI3K-p85from IL-1RI decreased the expressions of p-Akt, p-p70S6k and MAP2which increased by IL-1β stimulated (p<0.05, vs IL-1β group)Conclusion:1. This study successfully established a chronic MTLE model of immature rat, which was well coincidence with human chronic temporal epilepsy in seizure pattern, EEG recording and hippocampal pathology. High purity of astrocyte and neuron were also successfully cultured. The MTLE rat model and the primary astrocyte and neuron cells were prepared for the subsequent research to explore the developmental mechanism of MTLE from infancy to adult.2. MRP8, as inflammatory markers, activated the TLR4/NF-JκB/IL-1[signaling pathway to trigger the procession of pro-inflammatory factor release in MTLE pathological process, which provided important clues to elucidate the pathophysiological mechanisms of chronic MTLE and to identify an optimum medication intervention for the development of pharmacological therapies targeted at MTLE.3. IL-1β activated PI3K-p85through combining with IL-1RI to promote the activation and proliferation of neuron synapses via PI3K/Akt/mTOR signaling pathway, which might be one of the mechanisms of MTLE chronic progress. Inhibiting inflammatory reaction and its related signaling pathway may be an important direction for future research on refractory MTLE. |
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