| Status epilepticus(SE)is a severe medical emergency in clinic.The current treatment principle is to terminate seizures as soon as possible while administrating supportive treatment.About one-third of patients,however,suffer from unsuccessful control of seizures.SE of these patients develops into refractory status epilepticus(RSE).The longer the seizure lasts,the more harmful it will be to the patients.It is currently believed that continuous seizure activity will lead to inflammation,oxidative stress,electrolyte imbalance,acid-base imbalance,etc.which will cause neuronal death and finally result in cognitive impairment.Therefore,it has become urgent in clinic to solve the problems including how to terminate the seizure promptly,how to protect neurons and other cells,and how to improve the cognitive impairment caused by seizures.It has been found that molecular hydrogen can exert protective effects in many neurological disease models,such as models of cerebral infarction,Parkinson’s disease,and Alzheimer’s disease.Hydrogen can diffuse freely to cross blood-brain barrier,to penetrate the cell membrane and to diffuse into tissues and cells rapidly.In addition,as an antioxidant,it selectively reduces the most cytotoxic reactive oxygen species “·OH”.It is also reported that hydrogen shows anti-inflammatory effects and is able to regulate ion channel receptors.For example,hydrogen could alleviate hyperalgesia through inhibiting membrane transport of N-methyl-D-aspartate(NMDA)receptor subunit NR2 B,which in turn regulates the function of NMDA receptor and reduces the excitability of neurons.Because of the properties of hydrogen,we speculated that hydrogen may have a beneficial effect on RSE.Therefore,with the aim to provide novel theoterical and practical basis for solving RSE in clinical situations,we made attampt to demonstrate the role of hydrogen on RSE and to reveal the underlying mechanism.Objectives: 1.To examine the effect of hydrogen treatment on RSE and the effect on the cellular membrane expression and phosphorylation of NR2 B in the hippocampus.2.To explore the effect of hydrogen treatment on RSE-caused death of neural cells.3.To observe the effect of hydrogen treatment on cognitive impairment caused by RSE.Methods: 1.After the establishment of experimental model of RSE,we used behavior Racine scaleto observe the effect of hydrogen-rich saline(HRS)treatment on the severity ofepileptic seizures.Meanwhile,we monitored rat EEG activities and analyzed thechanges of amplitude and power spectrum after HRS treatment.Then,we usedWestern blot to examine the expression of NR2 B and p-NR2 B on the cell membraneof hippocampal tissues.2.By using immunofluorescence staining,transmission electron microscope and Westernblot,we examined the cell death modes and locations,and inflammation in thehippocampus of each group after HRS treatment.3.We employed Morris water maze to test the cognitive function of rats in each group 14days after RSE.Indicators including the escape latency,the duration of stay in thetarget quadrant and the frequency of entering the target quadrant were examined.Results: 1.Behavior Racine scores and EEG results.In Racine scores,there was no significant difference between RSE+saline group and RSE+HRS group.Compared with the rats in RSE+Saline group,the EEG amplitude and power of the rats in RSE+HRS group decreased.2.Changes in NR2 B expression and phosphorylation levels.Compared with the control(Con)group,the expression level and phosphorylation level of NR2 B on hippocampal cell membrane in rats of RSE group increased gradually as the seizure lasted.The expression level of NR2 B on hippocampal cell membrane was not significantly different between RSE+Saline group and RSE+HRS group,but the phosphorylation level of NR2 B was significantly lower in RSE+HRS group than in RSE+Saline group.3.Changes in apoptosis.Compared with Con group,apoptotic cells were significantly increased in CA1,CA3 and DG in RSE group.Compared with the RSE+Saline group,the percentage of apoptotic cells in RSE+HRS group in CA1 and DG decreased,while the percentage of apoptotic cells in CA3 did not significantly change.Furthermore,the cell type analysis of apoptosis showed that the percentage of apoptotic cells within neurons in CA1 in RSE+HRS group was significantly lower than that in RSE+Saline group,and the number of survived neurons in the CA1 region increased in RSE+HRS group than RSE+Saline group.Meanwhile,there was no significant difference of the number of the total and apoptotic astrocytes between RSE+HRS group and RSE+Saline group.In DG,the proportion of apoptotic cells within neurons and astrocytes was significantly lower in RSE+HRS group than in RSE+Saline group.4.Changes in necroptosis.(1)The morphological data revealed that the number of necroptotic cells in hippocampus of RSE group increased significantly when compared with Con group.The occurrence of necroptosis was further confirmed by Western blot and transmission electron microscopy results.Necroptosis was mainly distributed in CA1 and DG,and the necroptotic cells in CA1 were mainly neurons,while in DG they were mainly astrocytes.(2)Western blot analysis showed that in CA1 the expression of necroptosis-related protein,including MLKL,p-MLKL and RIP3,was significantly reduced in RSE+HRS group when compared with RSE+Saline group.(3)The staining results of CA1 region indicated that the number of MLKL positive cells within CA1 neurons was significantly increased in RSE+Saline group when compared with Con+Saline group and Con+HRS group.But it significantly dropped again in RSE+HRS group compared with RSE+Saline group.(4)The staining results of DG region indicated that the proportion of MLKL positive astrocytes within the astrocytes of DG region in RSE+Saline group and RSE+HRS group was significantly higher than in Con+Saline group and Con+HRS group,but the proportion significantly decreased in RSE+HRS group compared with RSE+Saline group.5.Changes in the activation of hippocampal microglia.The number of Iba1-positive microglia in the hippocampus of RSE+Saline group and RSE+HRS group was higher than that of Con+HRS group and Con+Saline group.However,compared with RSE+Saline group,the number of Iba1-positive hippocampal microglia in the RSE+HRS group significantly decreased.6.Changes in cognitive function of rats.Rats in RSE+Saline group and RSE+HRS group had significantly longer escape latency than those in Con+HRS group and the Con+Saline group,while the time they stayed in the target quadrant and the number of times they entered the target quadrant was significantly less than those in the Con+HRS group and the Con+Saline group.Compared with the RSE+Saline group,the escape latency of rats in the RSE+HRS group was significantly shortened,and the time they stayed in the target quadrant and the number of times they entered the target quadrant significantly increased.Conclusion: 1.HRS treatment had no effect on the behavioural Racine scores but can reduce the amplitude and power of EEG in RSE rats.The phosphorylation level of NR2 B subunits was reduced by HRS treatment.2.HRS treatment alleviated the RSE-induced cellular death.It could reduce the apoptosis and necroptosis of hippocampal neural cells induced by RSE,especially it could prevent the necroptosis of neurons in CA1 region.It could also significantly reduce the activation of microglia in the hippocampus.3.HRS treatment ameliorated cognitive impairment caused by RSE.4.HRS treatment not only attenuated abnormal EEG discharge in RSE rat,but also increased the number of survived cells in hippocampus.In addition,HRS treatment could improve the cognitive function of RSE rats.These results suggested that,clinically,alleviating abnormal EEG discharge might be beneficial for RSE patients. |
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