Objective: To study the expression of N-methl- D -aspartate (NMDA) receptor subunits NR2A and N R2B mRNA after audiogenic seizure in the brain of genetically epilepsy-prone ra t s-P77PMC. Methods: In situ hybridization technique was used to stud y the mRNA expre ssion of NR2A and NR2B at different times after audiogenic seizure in different b rain areas of P77PMC rats. Results: The NR2A mRNA expression showed time-depende nt down-regulation in cerebral motor and sensory cortex, hippocampus CA1-CA3, de ntate gyrus after audiogenic seizure. Down-regulation emerged 30 min after se izure, 2 hours later the mRNA reached the lowest level， 4-8 hours later re turned to the baseline, and 24 hours later decreased lower than baseline again . The mRNA expression of NR2B was quite similar to that of the NR2A af ter seizure. Conclusion: The down-regulation of NR2A and NR2B mRNA indicates that the subunit composition of the NMDA receptor complexes may be altered after aud iogenic seizure. These structural alterations could make the function of NMDA re ceptor complexes change and subsequently influence the excitability of brain as well as the susceptibility of epilepsy.

Epilepsy is one of the most common diseases in children.The diagnosis,classification and treatment of epilepsy are improving gradually and meet with the international standards.However,the understanding of the clinical significance of epilepsy combined with the mental and behavioral problems is not sufficient,which leads to the backlogging of the diagnosis and intervention of the co-morbidity,and further affects the long-term prognosis.The prevalence of epilepsy co-morbid with attention deficit hyperactivity disorder (ADHD) is high,which seriously affects the quality of the lives in children and their families.Timely diagnosis and standardized treatment is very important for the clinical workers to improve the long-term prognosis of these children.In this paper,the possible mechanism,the disease characteristics,the standardized diagnosis and treatment of epilepsy combined with ADHD,are briefly introduced.

Epilepsies associated with KCNQ2 are a spectrum of disorders,and KCNQ2 mutations lead to a variety of phenotypes from the severest to the mildest,from early onset epileptic encephalopathy (EOEE) to benign familial neonatal convulsion (BFNC).The type and the position of KCNQ2 mutations may be associated with phenotypes,and provide a clue for assessing prognosis.KCNQ2 mutations lead to the reduced protein expression,abnormal distribution of potassium channels or decreased currents of potassium channels.It is the potential mechanism that KCNQ2 mutations cause different phenotypes.Recent studies have also found that KCNQ2 mutations may trigger seizures by enhancing potassium channel currents;however it needs further studies to verify.

Objective:To improve previous method of primary rat cortical neuron culture to get purer and more long-lasting cells for study.Methods:Timed-pregnant Wistar rats at a gestational age of 16 or 17 days(16-17 d) were used.Fetal brains were removed and the cerebral cortices were dissected out.Papain digestion and mechanical dissociation were combined to conduct mono-cell suspending media.Four to six hours(4-6 h) post-plating,all plating media were removed from cultures and replaced with Neurobasal medium supplemented with B27.On the third day,10 ?mol/L cytosine arabinoside(Ara-C) was added to the culture for 24 h to inhibit the outgrowth of glial cells.Half of the culture medium was changed every week.The morphological changes of neuron cells were observed by light microscope.Double immuno-staining of microtubule-associated protein 2(MAP2) and karyon were applied to assess the culture purity.Evaluation of synapse formation was processed by immunocytochemical analysis using antibodies against both pre-and postsynaptic protein markers.Results:The improved method could remarkably increase the cell number and reduce neuronal damnification.The primary culture was characte-rized by high uniformity,purity,normal synapse formation and longtime livability.Conclusion:This is a simple and reliable technique for the in vitro primary culture of rat cortical neurons.

ObjectiveTo investigate the correlation of MRI features and phenotypes and genetic mutations in Pelizaeus-Merzbacher disease.Methods Sixteen boys with clinical diagnosis of PelizaeusMerzbacher disease (PMD) were included in this study.Their ages ranged from 22 months to 9 years.They were examined by pediatric neurologists,and clinical classification was made according to the symptoms and physical signs.An experienced radiologist reviewed the cranial MRI images and analyzed the brain involvement,including pallidus globus,pyramidal tract,corpus callosum,cerebellar white matter,semiovale centrum,brain atrophy and ‘ tigroid sign’.ResultsThere were 8 patients with classic form,7 patients with transitional form and one patient with connatal form.They all showed diffuse delayed myelination in the white matter,with involvement of pallidus globus in 13 cases,pyramidal tract in 7 cases,corpus callosum in 11 cases,cerebellar white matter in 7 cases,semiovale centrum in 12 cases.Cerebral atrophy was found in 5 patients and eerebellar atrophy was found in one patient.Five cases depicted ' tigroid sign'.In patients with PLP1 gene point mutation,pyramidal tract and cerebellar white matter involvement showed a high incidence.Cerebellar white matter lesions were relatively frequent in children with transitional form and connatal form.In contrast,‘ tigroid sign' was often related to classic form,which indicated a better myelination and outcome.ConclusionPMD patients show distinct imaging features in their brains,which may be correlated with the phenotype and genetic mutation.

Biomedical Technology ; Child ; Genetic Testing ; methods ; Humans ; Nervous System Diseases ; diagnosis ; genetics ; therapy ; Pediatrics ; Quality Improvement

Objective: To study the protection of IL 1ra in cultured developing neuron injury following Mg 2+ free induced epileptiform discharges. Methods: Rat embryo cortical neurons cultured for 6 d and 17 d were directly exposed to Mg 2+ free media, or pretreated with IL 1 receptor antagonist or NMDA receptor antagonists before being exposed to Mg 2+ free media, and then returned to regular media.MTT assay was used to study mitochondrial function injury, laser scanning confocal microscope to measure [Ca 2+ ]i, and real time RT PCR to detect gene mRNA expression. Results:(1) MTT conversion rates were higher in neurons pre and co treated with 10 mg/L IL 1ra than those of neurons with only Mg 2+ free treatment in neurons cultured for 17 d, but not in neurons cultured for 6 d.(2) [Ca 2+ ]i was lower in neurons pre and co treated with 10 mg/L IL 1ra than those of neurons with only Mg 2+ free treatment, either in neurons cultured for 6 d or in neurons cultured for 17 d, and the effects of IL 1ra on [Ca 2+ ]i change were different between neurons cultured for 6 d and neurons cultured for 17 d.(3) Pre and co treated with 10 mg/L IL 1ra NR1 mRNA expression increase induced by Mg 2+ free treatment was decreased, either in neurons cultured for 6 d or neurons cultured for 17 d, and this effect showed no difference between neurons cultured for 6 d and 17 d; Pre and co treated with 10 mg/L IL 1ra NR2A mRNA expression increase induced by Mg 2+ free treatment in neurons cultured for 17 d was decreased, and NR2A mRNA expression showed no difference between IL 1ra group and age matched control group, but have no effect on neurons cultured for 6 d; Pre and co treated with 10 mg/L IL 1ra have NR2B mRNA expression increase induced by Mg 2+ free treatment was not affected, either in neurons cultured for 6 d or neurons cultured for 17 d. Conclusion:Neuroprotection of IL 1Ra in seizure induced injury is age dependent. The mech anism of the neuroprotection of IL 1Ra includes down regulation of [Ca 2+ ]i and others.

Leukoencephalopathy with vanishing white matter(VWM) is one of the most prevalent inherited white matter disorders in childhood,and it′s the only known hereditary human disease due to the direct defects in protein synthesis process,with the gene defects in EIF2B1-5,encoding the five subunits of eukaryotic translation initiation factor(eIF2B ?,?,?,? and ?) respectively.eIF2B is essential for the protein translation initiation process,and its action is realized via eukaryotic translation initiation factor2(eIF2).Phosphorylation of eIF2? and eIF2B? is an important way to regulate eIF2B function,and thus play a key role in control of the protein translation level under physiological condition.Mutant eIF2B results in functional defects and decrease of the overall protein translation in cells,but in increase the translation of proteins with multiple upstream open reading frames,such as activating transcription factor 4(AFT4),which leads to the susceptibility to un-folded protein response under stress,and the following apoptosis.The exact pathogenic mechanisms of VWM are far from well understood.It′s suggested that level of AFT4 in cells with eIF2B mutations is higher than in wild type cells under physiological condition,which makes the mutant cells more susceptible to endoplasmic reticulum(ER) stress and unfolded protein response(UPR).Under stress,the defect eIF2B leads to a vicious cycle of UPR activation,which may underlie the neurological aggravation in VWM patients after minor stress,a specific cli-nical feature of VWM.Elucidating the pathogenesis of VWM will be helpful to further understand the protein translation process in eukaryotic cells,and provide a clue for possible therapeutic targets and treatment strategies in the future.Abstract:SUMM ARY Leukoencephalopathy with vanishing white matter(VWM) is one of the most prevalent in-herited white matter d isorders in childhood,and i′ts the only known hered itary human d isease due to the d irect defects in protein synthesis process,with the gene defects inEIF2B1-5,encod ing the five sub-units of eukaryotic translation initiation factor(eIF2B?,?,?,?and?) respectively.eIF2B is essential for the protein translation initiation process,and its action is realized via eukaryotic translation initiation factor2(eIF2).Phosphorylation of eIF2?and eIF2B?is an important way to regulate eIF2B function,and thus play a key role in control of the protein translation level under physiological cond ition.Mutant eIF2B results in functional defects and decrease of the overall protein translation in cells,but in increase the translation of proteins with multiple upstream open read ing frames,such as activating transcription factor 4(AFT4),which leads to the susceptibility to un-folded protein response under stress,and the following apoptosis.The exact pathogenic mechanisms ofVWM are far from well understood.I′ts sugges-ted that level ofAFT4 in cells with eIF2B mutations is higher than in wild type cells under physiological cond ition,which makes the mutant cellsmore susceptible to endoplasm ic reticulum(ER) stress and un-folded protein response(UPR).Under stress,the defect eIF2B leads to a vicious cycle ofUPR activa-tion,which may underlie the neurological aggravation in VWM patients afterm inor stress,a specific cli-nical feature ofVWM.E lucidating the pathogenesis ofVWM will be helpful to further understand the pro-tein translation process in eukaryotic cells,and provide a clue for possible therapeutic targets and treat-ment strategies in the future.

Institute of Pediatric,Suzhou University Affiliated Children's Hospital,Suzhou 215003,China Objective To explore the long-term effects of physical exercise on neonatal seizure-induced learning,memory deficit and the expression of zinc transporter-3(ZnT3)in rat hippocampus.Methods Sprague Dawlev rats aged 6 days were randomly divided into a recurrent-seizure group(RS)and a control group. At postnatal dav 6(P6),the recurrent seizures were induced by inhalation of the volatile agent flurothyl once a day for consecu tive 6 davs.The rats in the control group were placed in the container for an equal period of time as those in the RS group without exposure to flurothyl. Y-maze test was performed to evaluate learning and memory capacity at postnatal day 29 to 35 and 61 to 67,respectively.During the period of postnatal day 51 to 56,all the animals in the RS andcontrol groups were subject to a 30-minute daily aerobic exercise program for consecutive 6 days.All the animals weresacrificed at postnatal day 78,and the in situ hybridization method was used to detect the expression of ZnT3 mRNA in hippocampus. Results ①The number of trials needed for getting correct response to the electric stimulation in the first Y-maze test was(60±14.1)and(37.5±17.2)for the RS and control groups,respectively(P＜0.05),while that in the second Y-maze test carried out 24 hours later was(27.5±14.1)and(21±11.01)for the RS and the control groups,respectively(P＞0.05).②Memory test revealed no significant difference between the RS and thecontrol groups(P＞0.05).③In situ hybridization detection showed that the expression of ZnT3 mRNA in hippocam pus was not significantly different between the two groups.However,there showed a significant difference between the dentate gyrus and CA3 in the RS group with regard to the expression of ZnT3 mRNA(P＜0.05). Conclusions Physical exercise improves the learning capacity of neonatal seizure-induced cognitive deficit and might have effects on the regulation of zinc transporter gene expressions in hippocampus.

Objective To analyze and determine the clinical, molecular pathology and genetic features of a Chinese family with dystrophinopathy. Methods Clinical data of the proband and his family members were collected. Immunohistochemistry staining was performed on muscular biopsy tissues with antimerosin, emerin and the N, C and central rod domains of dystrophin. Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes. Multiplex ligation-dependent probe amplification (MLPA) was used to test Duchenne muscular dystrophy (DMD) gene to determine the ways and sites of genetic mutation, and analyze the relationships between genotype and phenotype. Results Patients from this family were clinically diagnosed as muscular dystrophy, and they presented serious manifestations although the immunohistochemistry analysis for the proband exhibited partial loss of dystrophin staining, and positive expression with merosin and emerin. Further test with MLPA detected the loss of exons 45--54 in DMD gene in the proband, while his mother had heterozygositic loss in exons 45--54. Conclusions The losses of exons 45--54 in the proband are all derived from his mother, who carries genetic mutation with normal phenotype. He has been diagnosed as dystrophinopathy. At the same time, his partial loss of dystrophin is not parallel to the out-of-frame mutation of the gene and his severe clinical manifestations. Abnormal expression of dystrophin is the pathological basis for dystrophinopathy phenotype. Its clinical outcome depends not only on the degree of the protein expression, but also on the function of the sites where the DMD gene less occurs.