Objective To investigate the location of CYLD in the neurons and explore the expression of CYLD in OGD/reperfusion-induced neuronal necroptosis.Methods Primary cortical neurons were cultured for 6days and neuronal purity was observed by double staining immunofluorescence of β3-tubulin and DAPI.The location of CYLD was identified by double staining immunofluorescence of NeuN,DAPI and CYLD using primary cortical neurons cultured for 14 days.Then,primary cortical neurons were divided into 8 groups:Control,EBSS,DMSO,OGD/reperfusion(0 h,2 h,6 h,8 h,12 h).Neurons were pretreated with zVAD-fmk for 30 min,OGD for 2 h and the levels of CYLD were evaluated after reoxygenation at different time points.The peak value(8 h) was chosen as reoxygenation time point.Neurons were divided into two groups as Control and OGD.The levels of CYLD were determined in both cytoplasm and nucleus after OGD 2 h and reoxygenation 8 h.Results The double staining immunofluorescence showed that neuronal cultured purity was about 70％ and the CYLD strongly expressed in nucleus but weakly in cytoplasm.The levels of CYLD increased gradually with different reoxygenation time and arrived at peak value after reoxygenation for 8 h (P ＜ 0.05),which was in accordance with the change of LDH (P ＜0.05) (Control (1.00±0.00),EBSS (1.07 ±0.03),DMSO (1.09 ±0.03),0h (1.40±0.12),2 h (1.74±0.08),6 h (2.25 ± 0.12),8 h (2.97 ± 0.15),12 h (3.01 ± 0.08)).The level of cytoplasm CYLD increased significantly in the OGD group (reoxygenation for 8 h)than that in control group (P＜0.05).But the level of nucleus CYLD had no difference between OGD and control group (P ＞ 0.05),which was in accordance with the results of immunofluorescence.Conclusion The CYLD in neurons cytoplasm is involved in necroptosis induced by OGD/deprivation and downregulating of CYLD has a protective effect on the brain injury resulted from ischemia/ reperfusion.

Objective To explore the value of Montreal Cognitive Assessment (MoCA) in the diagnosis of mild cognitive impairment. Methods Mini-mental state examination (MMSE) was used in 532 elderly persons aged 60 years and over in Xuzhou city Gulou county. The 69 healthy people and patients with mild cognitive impairment were chosen to undergo MoCA. Then the sensitivity and specificity of MoCA were analyzed. Results According to MMSE, there were 19 patients with mild cognitive impairment (27. 5 %), 50 healthy persons (72.5 %). While according to MoCA, there were 58 patients with mild cognitive impairment (84.1%), 11 healthy persons (15.9 %). The consistency of the two scales was not good. And compared with MMSE, the sensitivity of MoCA was 94.7%, and the specificity was 20. 1%. Conclusions In the diagnosis of mild cognitive impairment, MoCA is more sensitive than MMSE.

Objective To investigate anxiety of clinicians in tertiary hospitals,and discuss the fac-tors that influence clinicians'anxiety in order to provide reference for further effective intervention measures. Methods 427 clinicians from various departments in tertiary hospitals selected by cluster sampling method were investigate by self-rating anxiety scale ( SAS) .Statistical software SPSS 17.0 was used to analyze data for multivariate logistic regression analysis of its relevant influencing factors.Results 38.2%of the clini-cians had anxiety symptoms (55.0%was mild anxiety,26.1%was moderate anxiety,18.9%was severe anxi-ety) .Factors like education (χ2=7.3, P=0.03) ,job title (χ2=53.24, P<0.01) ,divisions (χ2=46.46, P<0.01) ,work experience (χ2=9.78, P=0.04) ,work overtime (χ2=4.29, P=0.03) ,worry of medical mal-practice (χ2=9.54, P<0.01),doctor-patient relationship tension (χ2=7.50, P<0.01),fear for personal safety (χ2=10.27, P<0.01) ,job promotion competition (χ2=11.40, P<0.01) ,labor and payment situation (χ2=6.36, P=0.01) ,colleagues'understanding and support (χ2=15.77, P=0.01) ,work environment (χ2=21.85, P<0.01) and leaders'attention (χ2=7.17, P=0.01) had significant differences between anxiety and non-anxiety clinicians.Multivariate logistic regression analysis revealed that factors like divisions ( com-pared with internal medicine,OB&GYNβ=0.98,surgeryβ=0.95,pediatricsβ=0.86,emergencyβ=0.69) , work overtime (β=0.18),fear for personal safety (β=0.86),fear of medical malpractice (β=0.79) and doctor-patient relationship tension (β=0.28) had a negative impact on anxiety,while factors like education ( compared with bachelors,mastersβ=-1.38,doctorsβ=-1.65) ,job title ( compared with primary,intermedi-ateβ=-0.33,highβ=-0.45,advancedβ=-0.59) ,work experience (β=-0.18 ) and work environment (β=-0.32) were protective factors.Conclusion There is a certain degree of anxiety among clinicians in tertiary hospitals,resulting in a heavy psychological burden on them.Active measures should be taken to improve the clinicians'psychological health.

BACKGROUND: Mild hypothermia (28-35 ℃) is becoming one of the promising methods in treating acute ischemic stroke. Hypothermia can effectively lessen brain edema, which is one of its neuroprotective roles.OBJECTIVE: To investigate the effect of mild hypothermia on brain water content and aquaporin-4 (AQP4) expression level following global cerebral ischemia-reperfusion injury in rats, so as to study the neuroprotective mechanisms of mild hypothermia.DESIGN: A randomized controlled trial.SETTING: Neurobiological Laboratory of Xuzhou Medical College.MATERIALS: 110 healthy male SD rats with body mass 250-300 g, provided by the Animal Center of Xuzhou Medical College, No. SYNK (Jiangsu) 2002-0079, were selected and randomly divided into 3 groups by SPSS 11.0software: ①sham-operated group (n=10);②normothermiagroup (n=50); ③mild hypothermia group (33 ℃, n=50). Normothermia group and mild hypothermia group were subdivided into five reperfusion subgroups for 6 hours, 1, 2, 3 and 7 days, respectively with 10 rats in each subgroup,in which 5 rats were used for measurement of brain water content, and others for HE staining and immunohistochemistry staining.METHODS: The models of global cerebral ischemia were established in the normothermia group and mild hypothermia group by four-vessel occlusion (4-VO) method with ischemia for 15 minutes as Pulsinelli described.The rats in the sham-operated group were only underwent the electrocauterization of bilateral vertebral arteries and the isolation of common carotid arteries except for occlusion of common carotid arteries. Twenty-four hours later, the rats were decapitated to take out the brains. The brains of rats in the normothermia group and mild hypothermia group were taken out to make sections for HE staining and immunohistochemistry staining, and the dynamic change of pathology of the brain tissue and AQP4 expression level were observed after reperfusion for 6 hours, 1, 2, 3 and 7 days, respectively. The brain wet-to-dry weight measurement was used to measure the brain water content of the rats at each time point of each group.MAIN OUTCOME MEASURES: ①The pathologic changes of brain tissues of rats in the normothermia group and mild hypothermia group. ②The brain water content and the AQP4 expression level of all rats at each time point.RESULTS: ①After 6 hours of reperfusion, brain edema appeared in the normothermia group including amplification of periyascular spaces and intercellular spaces, rarefaction of brain tissues, etc, which got worst after 2 days of reperfusion; the phenomenon of brain edema of the rats in the mild hypothermia group at each time point was relatively lighter than the normothermia group. ②Brain water content of the normothermia group and mild hypothermia group was increased after 6 hours of reperfusion and reached peak at the 2nd day; Although decreased at the 7th day, it was still higher than the sham-operated group. The brain water content of the mild hypothermia group at each time point was less than the normothermia group (values after 6 hour and 7 day, P ＜ 0.01, the rest groups P ＜ 0.05).③AQP4 expression level of the normothermia group and mild hypothermia group was increased after 6 hours of reperfusion and reached peak at the 2nd day. Although decreased at the 7th day, it was still higher than the sham-operated group.The AQP4 expression level of the mild hypothermia group at each time point was lower than the normothermia group (P ＜ 0.01).CONCLUSION:The change tendency of AQP4 level is parallel to that of brain water content after ischemia reperfusion, which indicates that the upregulation of AQP4 expression is one of molecular mechanisms for the for mation of ischemicbrain edema. Mild hypothermia can release ischemic brain edema by inhibiting AQP4 expression, which is one of its mechanisms.

ObjectiveTo evaluate the value of early secretory antigenic target 6 (ESAT-6) in monocytes of cerebrospinal fluid (CSF) in the early diagnosis and judgment of the tuberculous meningitis (TBM). MethodsForty cases with TBM (TBM group) and 40 patients with other meningitis patients (control group,9 cases with purulent meningitis,24 cases with viral meningitis,3 cases with cryptococcal meningitis,3 cases with cerebral cysticercosis, 1 case with neurosyphyilis) were involved in this study.Conventional examination of CSF was inspected after admission, and the ESAT-6 in monocytes of CSF was detected by immunocytochemical stain. ResultsThe positive rate of ESAT-6 was 24％ in TBM group and 0 in control group (P ＜ 0.01 ). In ROC curve, if the rate of ESAT-6 ≥8.5％ judged as positive, and ＜ 8.5％ judged as negative, 35 patients of TBM group and 4 patients of control group were ESAT-6 positive. The sensitivity of the ESAT-6 in the diagnosis of TBM was 87.5％, and the specificity of TBM was 90.0％. Thirty patients in TBM group were not given anti-tuberculosis drug treatment, and the positive rate of ESAT-6 was 93.3％ (28/30) before treatment, and was 46.7％ (14/30) after treatment of 2 weeks (P =0.000).ConclusionsESAT-6 in monocytes of CSF detected by immunocytochemical stain has higher sensitivity and specificity in the diagnosis of TBM in early stage. Observing the positive rate of ESAT-6 dynamicly has practical value in judgment the severity of TBM.

BACKGROUND:Psychiatric patients are the high risk group of people who are liable to commit suicide.Recently,research on neurobiology and genetics of suicide mainly concentrates in the study of 5 hydroxytryptamine(5 HT) system.It is found that T102C polymorphism of 5 HT2A receptor gene is associated with suicide in depressive patients. OBJECTIVE:To investigate the correlation of A 1438G polymorphism of 5 HT2A receptor gene with attempted suicide in psychiatric patients. DESIGN:An observational comparative study of taking the patients as subjects and healthy physical examinee as the controls. SETTINGS:A psychiatric department in a municipal hospital and a neurological department in a university hospital. PARTICIPANTS:All the samples in the research were collected from the Department of Psychiatry of Yangzhou Wutaishan Hospital from March to September 2002.Analysis of genotype was completed in the Neurobiological Laboratory of Xuzhou Medical Institute from October to December 2002.Inclusive criterion:patients met the diagnostic criteria of schizophrenia or affective disorder in the third edition of the Chinese Classification and diagnostic criteria of Mental Disorders(CCMD 3) and Diagnostic and Statistical Manual of Mental Disorders Fourth Edition(DSM IV).Exclusive criterion: parasuicider with idea or behavior of hurting oneself,but without intention to die.According to whether there was suicidal behavior or intention,116 patients were divided into 2 groups:attempted suicide group[n=52,35 males and 17 females,with an average age of (44± 13) years old] and no suicide group [n=64, 44 males and 20 females, with an average age of (48± 15) years old].Other 63 cases were taken as the normal controls[33 males and 30 females, with an average age of (55± 17) years old]. INTERVENTIONS:DNA of all the participants was extracted with the routine method of chloroform saturatedphenol leukocyte pheresis.The polymorphism of 5 HT2A receptor gene was analyzed with the restriction fragment length polymorphism. RESULTS:The G allele frequency of A 1438G polymorphism in the attempted suicide group(0.52) was significantly higher than that in the normal control group(0.39)(χ 2=3.91,P< 0.05).There were significant differences in the distribution of the 3 genotypes of AA, AG and GG between the attempted suicide group(0.23,0.50, 0.27) and normal control group(0.32, 0.59, 0.09)(χ 2=6.12,P< 0.05). CONCLUSION:Attempted suicide of psychiatric patient is associated with A 1438G polymorphism of 5 HT2A receptor gene.G allele may be a risk factor for the attempted suicide of psychiatric patient.

Objective To investigate the location of receptor interacting protein 3( RIP3) in Necroptosis and its function in this signal passage, and explore the relationship between receptor interacting protein 1 ( RIP1 ) and RIP3 in nuclear translocation. Methods Primary cerebrocortical neurons were cultured for 12 days,then pre-treated with zVAD-fmk(20μ,mol/L) for half an hour to block apoptosis. ①Extracting nuclear and cytoplasmic protein after neurons were exposed to TNF for different time ,then protein levels of RIP3 were analyzed by western blot and immunofluorescence for qualitative observation;②In the following research,the neurons were treated with Nec-1 and shRlPl ,then the protein level of RIP1 and RIP3 with western blot were analyzed, cell viability were determined by measuring LDH levels. Results ①In signaling pathways of necroptosis, the protein level of RIP3 in cytoplasmic decreased gradually with prolonged TNF exposure, to the corresponding it rolled up in nucleus and a-chieved the peak in 12 hours of TNF treatment ( Cytoplasmic 0. 45 ± 0. 03 ,0. 41 ± 0. 02,0. 73 ± 0. 03 ,0. 90 ± 0.01,1.15 ±0.04,1.30 ±0.02,0.99 ±0.03,0.63 ±0. 03;Nucleus 0. 07 ±0.02,0. 26 ±0.02,0. 57 ±0. 02,0. 68 ± 0.02,0. 80 ± 0.01,0.92 ± 0.02,1.28 ± 0.03,0. 87 ± 0.02) (P < 0.01). ②Blocking the relationship between RIP1 and RIP3 with necrostatin-1 and shRIPl , nuclear translocation of RIP3 decreased and caused a great increase in cell viability( 1.00 ±0.05,0.39 ±0.03,0.50 ±0. 03) (P<0. 01). Conclusion RIP3 mainly locates in cy-tolymph of normal cells,it translocates into nucelus as necroptosis takes place. RIP1 function with RIP3 in nuclear translocation. Block nuclear translocation of RIP3 is a potential way to protect cells.

10.3969/j.issn.2095-4344.2013.23.018

Objective To observe the effect of electroacupuncture (EA) plus oxygenmedicine (OM) on the expression of Bcl-2 and Bax in the hippocampal CA 1 area in cerebral ischemia/reperfusion injury (CI/RI) rats. Methods Thirty SD rats were randomized into sham-operation,model,EA,OM,EA+OM groups (n=6/group). CI/RI model was established by using modified Pulsinelli 4 vessel occlusion and reperfusion. EA (100 Hz,3.5 mA) was applied to "Baihui" (GV 20) and "Zusanli" (ST 36) 30 min,once daily for 4 days. Rats of OM and EA+OM groups were put into a box filled with oxygen and atomized herbal medicines containing Bingpian (Borneolum),Shexiang (Moschus),Huangjing (Rhizoma Polygonati),Shouwu (Radix Polygoni Multiflori),etc. for 30 min,once daily for 4 days. Bcl-2 and Bax expression of the hippocampal CA 1 area was detected by immunohistochemistry. Results Compared with sham group,the numbers of Bcl-2 immunoreaction (IR) and Bax IR positive cells,and the immunoactivity of Bcl-2 IR and Bax IR positive products in the hippocampal CA 1 area were increased significantly in model group (P0.05). Conclusion EA and OM and EA+OM can effectively regulate the expression of Bcl-2 and Bax in the hippocampal CA 1 area in CI/RI rats,and the effects of EA+OM are significantly superior to those of simple EA and simple OM,which may contribute to their effect in improving cerebral ischemia.

Objective To explore the effect of L-dopa on plasma homocysteine and folic acid in patients with parkinsons' s disease(PD).Methods Twenty eight elderly PD patients and thirty normal subjects were enrolled in this group.The homocysteine,cobalamin and folate were examined in normal group and in PD group before treatment and after being treated with L-dopa for six moths respectively.Then the homocysteine,cobalamin and folate were compared between the two groups.Results The plasma homocysteine levels increased in PD patient group after being treated with L-dopa for six months(19.19? 8.01)?mol/L as compared with those of the PD group before treatment(12.50?3.78)?mol/L and those of control group(12.60?3.94)?mol/L(P