| Background and ObjectiveEpilepsy is a common disease which affects human health severally in body and mental, and has a morbidity rate at 7.5‰ in lifetime. In clinic, seizure can be controlled by anti-epilepsy drugs(AEDs), but there is 20-30% patients poorly controlled and become refractory epilepsy eventually. Neurosurgery could save 50% patients with refractory epilepsy; however, AEDs are still in need. There is a task in neurology on finding new mechanism related to epilepsy and AEDs.D-serine has a role similar to neurotransmitter, which can be released by synaptic vessel in glia, and regulates neural activity combining with NMDA receptor. D-serine plays a role in pathomechanism of many neurological diseases, including amyotrophic lateral sclerosis(ALS), schizophrenia, Alzheimer disease, epilepsy and so on. Metabolism of D-serine mainly relays on serine racemese(SR) and D-amino-acid oxidase(DAAO). SR predominantly locates in astrocyte and neuron, which could transform L-serine into D-serine. The distribution of SR could be a marker presenting distribution and function of D-serine. Based on the known, we find there is an antiseizure effect related to the antagonist of the glycine binding site of NMDA receptor. Meanwhile, the synthesis of D-serine is increasing in mice brain, correlating to the phosphorylation of NMDA receptor. In all, we set a hypothesis that the attack of seizure possibly come with the increase of SR in astrocyte, and the synthesis and secretion of D-serine in astrocyte is up leveled, overexpressed D-serine may play a role in seizure by activing NMDA receptor. In all, we explore the effect and mechanism of D-serine in epilepsy by the rat lithium-plocarpine model to finding potential target for epilepsy patients. Material and Methods1)Immunohistochemistry method: observe the character of SR in normal young and adult rat hippocampal slices, in advance, we research the distribution of SR in epilepsy patients compared with traumatic brain injury ones.2) Fixing lateral ventricles cannula and hippocampal electrodes, we make lithium-pilocarpine model 30 minutes later for preconditioning inhibitor(SR inhibitor, DAAO inhibitor, saline as control) injecting into lateral ventricles and NMDA receptor inhibitor intraperitoneal injecting.3) Lithium-pilocarpine model is done one week later after fixing lateral ventricles cannula and hippocampal electrodes.4) After video electroencephalogram monitor, we choose the rat to prepare brain cryosections.5) After making protein of brain tissues, we explore the level of AKT/P-AKT, ERK/P-ERK, JNK/P-JNK signal molecules in rat brain by the measurement of gray scale in Western blot; meanwhile, we detect SR/GFAP and check out the positive numbers in brain cryosections by immunoflorescence. Results1) The location and distribution of SR in young and adult rats and the hippocampal specimen of epilepsy patients: SR locates mainly in neuron and astrocyte. In hippocampal CA areas, the quantity of SR positive cells in adult rats is more than youngs, conversely, the number of positive glia is more in youngs than adult rats. Compared with traumatic brain injury group, there are obviously more SR positive cells in hippocampal specimen of patients with refractory epilepsy.2) Interfering the level of D-serine has effects on the change of behavior and EEG(electroencephalogram) in rat pilocarpine model. In our research, we discuss the difference between pilocarpine control group, MK801(generous NMDA receptor inhibitor) group, LaaβH(SR) group and CBIO(DAAO inhibitor) group. The result demonstrates that, compared with pilocarpine control group, inhibiting the activity of SR can prolong the latent period of Racine 3 level and SE, meanwhile, the total EEG power is lower, and the total EEG power of MK801 group is decreased dramatically.3) Interfering the level of D-serine has influence on the expression and activity of ERK signal molecular: comparing with control group, inhibiting activity of SR induces the dramatic reduction of P-ERK, however, downing the activity of DAAO cause the significantly increasing of P-ERK level. In all, it reminds us that the increasing of D-serine may be an important factor in activity of ERK signal. Conclusion1)SR is distributing in hippocampal neuron and astrocyte in both rats and epilepsy patients, the quantity and distribution of SR are correlating with age and the condition of epilepsy.2)Inhibition of the synthesis of SR in brain could delay the latent period of Raccine 3 level and SE in rats, as same as decrease the total EEG power in SE.3)D-serine may involve in the pathology of epileptic seizure by the path of activing ERK signal pathway.In summary, this study suggests that the abnormal change in level of D-serine in brain may involve the mechanism of epileptic seizure; it’s possible that the intervention of D-serine metabolism be a potential target for epilepsy treatment, and our teamwork provide the primary evidence for epilepsy cure. |
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