ABSTRACT:Objective To investigate changes in the neutrophils in rats with D-galactosamine (D-GalN)-induced acute liver failure (ALF)and to explore the therapeutic effect of interventions treatment of neutrophils on ALF.Methods Liver function,the expressions of inflammatory cytokines TNF-αand IL-1β,and the changes of neutrophils in the peripheral blood and the liver were observed in rats with D-GalN (intraperitoneal injection)-induced ALF.SD rats were randomly divided into three groups when treated with intervention of neutrophils:control group,ALF group (intraperitoneal injection of D-GalN),and treatment group (intravenous injection of anti-PMN serum via tail vein 24 h before modeling).Biochemical analysis was used to detect serum ALT,AST, TBIL and blood ammonia.Hematology analyzer was applied to analyze the number and percentage of peripheral blood neutrophils.The number of neutrophils in the liver was evaluated by immunohistochemistry.Liver RT-PCR was adopted to detect the mRNA expression of inflammatory cytokines TNF-αand IL-1β.Results We found that 6 h after D-GalN injection,serum ALT,AST,TBIL and blood ammonia in ALF rats were significantly increased (P <0.05).The mRNA expression levels of inflammatory cytokines TNF-αand IL-1βin the liver reached the peak at 6 h after modeling (P <0.001),and it was still notably higher at 24 h than before modeling (P <0.001 ).The number and percentage of peripheral blood neutrophils and the number of neutrophils in the liver were all markedly increased 12 h after modeling (P <0.001 ),and the increase continued at least until 24 h (P <0.001 ).24 h after intravenous injection of anti-PMN serum via tail vein,ALF rats had a distinct decrease in the number of peripheral blood neutrophils and neutrophils in the liver 24 h after modeling (P <0.001).Meanwhile,serum ALT,AST,TBIL and blood ammonia were all greatly decreased compared with those in ALF group (P <0.05);a significant reduction of hepatocyte apoptosis was observed.Also,the expressions of TNF-α and IL-1β in the liver were remarkably decreased after treatment (P <0.05).Conclusion Neutrophils accumulated in peripheral blood and liver of rats with D-GalN-induced ALF.The treatment of anti-PMN serum may have a therapeutic effect on liver function and immune microenvironment in ALF rats.

ObjectiveTo investigate the efficacy of different methods of anesthesia for laparoscopic hysterectomy.MethodsSixty ASA Ⅰ or Ⅱ patients,aged 45-60 yr,weighing 55-65 kg,scheduled for laparoscopic hysterectomy,were equally and randomly divided into 2 groups:combined intravenous-inhalational anesthesia group (group Ⅰ ) and combined spinal-epidoral anesthesia (CSEA) + general anesthesia group (group Ⅱ ).In group Ⅰ,anesthesia was maintained with inhalation of sevoflurane and infusion of remifentanil after induction of anesthesia.In group Ⅱ,CSEA was performed,after the upper level of sensory block was stable,general anesthesia was induced and maintained with inhalation of sevoflurane,and state entropy (SE) was naintained at 45-60.Arterial blood samples were taken to determine the plasma concentrations of adrenaline ( AE ),norepinephrine (NE) and dopamine (DA) after admission to the operation room,after completion of pneumoperitoneum,at 10 min after pneumopentoneum,during uterus traction,during removal of the laryngeal mask airway,and at 10 min after removal of the laryngeal mask airway (T0-5).The time for recovery of spontaneous breathing,extubation time,and time of regaining consciousness were recorded at the end of operation.The side-effects and number of patients requiring increments of analgesics were also recorded within 48 h after operation.Patient' s satisfaction was recorded at 48 h after operation.ResultsCompared with group Ⅰ,the plasma concentrations of AE and NE at T3-5 and the plasma concentrations of DA at T3,5 were significantly decreased,the time for recovery of spontaneous breathing,extubation time,and time of regaining corsciousess were significantly shortened,and the incidence of agitation and the number of patients requiring increments of analgesics were significantly decreased in group Ⅱ ( P ＜0.05).There was no significant difference in the incidence of intraoperative awareness,and nausea and vomiting after operation,and the level of patient' s satisfaction at 48 h after operation between the two groups ( P ＞ 0.05).ConctusionCSEA + general anesthesia has better efficacy than combined intravenous-inhalational anesthesia when used for laparoscopic hysterectomy.

Objective To evaluate the effect of dexmedetomidine on left ventricular function in patients undergoing coronary artery bypass grafting (CABG) by transesophageal echocardiography (TEE).Methods The study was a prospective,randomized and placebo-control clinical trial.Eighty patients undergoing CABG with cardiopulmonary bypass (CPB) were divided into dexmedetomidine group (group D)and control group (group C) by random digits table method with 40 cases each.A loading dose of dexmedetomidine 0.5 μg/kg was injected intravenously 10 min after induction followed by infusion at 0.4 μ g/(kg· h) until the end of operation in group D,while equal volume of normal saline was given in group C.Left ventricular function was assessed by transesophageal echocardiography before the infusion of dexmedetomidine (T1),at the end of the infusion of loading dose (T2),before CPB (T3) and at the end of the operation (T4).Results Compared with those at T1,left ventricular ejection fraction and fractional area change decreased significantly [(58.0 ± 12.0)％,(60.0 ± 9.6)％ vs.(63.0 ± 8.6)％ and (46.0 ± 9.3)％,(48.0 ± 8.4)％ vs.(51.0 ± 6.7)％] (P ＜ 0.05 or ＜ 0.01),E/A ratio increased significantly (1.05 ± 0.27,1.07 ±0.31 vs.0.98 ±0.19)(P ＜0.05 or ＜0.01) and myocardial performance index (MPI) decreased significantly (0.46 ± 0.14,0.45 ± 0.12 vs.0.51 ± 0.14) (P ＜ 0.05) at T2 and T3 in group D,while stroke volume was not significantly changed (P＞ 0.05).Compared with that in group C,E/A ratio and rapid filling fraction in group D was significantly higher [1.06 ± 0.18 vs.0.97 ± 0.18,(62.0 ± 7.1)％ vs.(58.0 ± 7.3)％],and S/D ratio and MPI was significantly lower at T4(1.17 ± 0.21 vs.1.29 ± 0.22,0.43 ± 0.15 vs.0.50 ± 0.15),and there were significant differences (P ＜ 0.05).There was no difference in the parameters indicating left ventricular systolic function (P ＞ 0.05).Conclusions Dexmedetomidine restrains left ventricular systolic function in the patients undergoing CABG,but does not decrease the cardiac output,and improve relaxation dysfunction of left ventricular diastolic function.Global left ventricular function is improved by dexmedetomidine after CABG.

OBJECTIVETo study the effect of electroacupuncture on nitric oxide synthase (NOS) in rats with cerebral ischemia-reperfusion injury.

METHODSFocal cerebral ischemia-reperfusion model was established using modified intravascular suture technique. The NO content in the brain tissue was detected by nitrite reduction and the expressions of nNOS and iNOS were detected by immunohistochemistry. Eighty rats in this experiment were divided into the normal group, the cerebral ischemia-reperfusion injury model group (as the model group), the cerebral ischemia-reperfusion injury + electroacupuncture group (as the acupuncture group), and the cerebral ischemia-reperfusion injury + phosphatidylinositol 3 kinase (PI3-K) inhibitor group (as the inhibitor group). Each group consisted of twenty rats. Five microL PI3-K inhibitor LY294002 (400 microL) was slowly injected at the lateral cerebral ventricle of rats in the inhibitor group at a constant speed using microinjector according to Konig Klippel atlas of the stereotaxis instrument. Shuigou (DU26) and Chengjiang (RN24) were selected to determine levels of NO and NOS.

RESULTSAfter 24-h ischemia-reperfusion, the NO levels of the hippocampus and the cerebral cortex increased abnormally, and the expressions of nNOS and iNOS increased, showing significant difference when compared with those of the normal group (P<0.05). By electroacupuncture at Shuigou (DU26) and Chengjiang (RN24), the ischemic cerebral ischemia-reperfusion injury neuron loss was inhibited. Meanwhile, the high levels of NO, nNOS and iNOS in the cerebral cortex and the hippocampus were significantly inhibited (P<0.05). The abnormally increased expressions of nNOS and iNOS were reversed, showing significant difference when compared with the model group (P<0.05). But when compared with the normal group, there was no significant difference (P>0.05). The effects of electroacupuncture reversed the abnormally increased NO levels of the hippocampus and the cerebral cortex and expressions of nNOS and iNOS after LY294002 oppressed anti-PI3K to block the TrkA acceptor circuit. The NO levels of the hippocampus and the cerebral cortex and expressions of nNOS and iNOS increased again, showing significant difference when compared with the acupuncture group (P<0.05).

CONCLUSIONSAcupuncture fought against cerebral ischemia and reperfusion in the loss of neurons, at the same time, the abnormal regulation of NOS had reverse effect partly through TrkA/PI3K mediated signal transduction pathway.

Animals ; Brain Ischemia ; metabolism ; Electroacupuncture ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type I ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; Signal Transduction

Background and purpose:In 2013, the ofifcial journal of European Society of Radiotherapy &Oncology (ESTRO) -Radiotherapy & Oncology published the updated version of Consensus Guidelines of Delineation of the neck node levels for head and neck tumors, which contributed to the standardization of description of neck nodal metastasis, as well as reduction of treatment variations from various institutions. This study applied this updated guidelines to analyze the patterns of lymph node metastasis of nasopharyngeal carcinoma and explore the prognostic value of the radiologic characteristics of nodes, in order to provide evidence for future revision of N staging system. Methods:A total of 656 patients from Jan. 2009 to Dec. 2010 were retrospectively recruited to analysis. All were pathologically diagnosed as non-metastatic nasopharyngeal carcinoma, treated with intensity-modulated radiotherapy. All patients received a pretreatment MRI scan. We retrospectively reviewed the MRI imaging of 656 patients and mapped the lymph node metastasis using the 2013 International Consensus Guidelines.Results:Median follow-up was 46.9 months. Four-year local recurrence-free survival, nodal recurrence-free survival, distant metastasis-free survival, disease-free survival and overall survival was 91.3%, 95.1%, 87.7%, 78.5% and 92.8%, respectively. The most common metastatic node levels were levelⅡ (76.2%) and levelⅦa (65.1%), followed by levelⅢ (50.4%),Ⅴa(17.5%) andⅣa (11.7%). There was a very low incidence of node skipping (1.0%). Cervical nodal necrosis was observed in 46.4%of patients with positive nodes and extracapsular spread was noted in 74.4% of them. Univariate analysis showed that bilateral nodal involvement, greatest dimension of positive nodes (≥6 cm), central nodal necrosis, T stage and N stage were prognostic factors for disease-free survival and distant metastasis-free survival (P<0.05). Extracapsular spread showed a trend to correlate with poor distant metastasis-free survival (P=0.060). The involvement of lower neck levels (below the caudal border of cricoid cartilage) did not have a signiifcant impact on disease-free survival and distant metastasis-free survival. In multivariate analysis, T stage and greatest dimension of nodes (≥6 cm) were independent prognostic factors for distant metastasis-free survival (P<0.05). T stage, greatest dimension of nodes (≥6 cm) and central nodal necrosis were independent prognostic factors of disease-free survival (P<0.05).Conclusion:This study demonstrates the patterns of lymph node metastasis of nasopharyngeal carcinoma based on 2013 International Consensus Guidelines. Bilateral nodal involvement, greatest dimension of positive nodes and central nodal necrosis had prognostic values on disease-free survival and distant metastasis-free survival. In our study, the involvement of lower neck levels was not proved to be a prognostic factor for disease-free survival and distant metastasis-free survival.

Objective To evaluate the effect of acute plateletpheresis (APP) and back-transfusion on platelet activation in the patients undergoing open heart surgery with cardiopulmonary bypass (CPB).Methods Forty patients,aged 35-64 yr,with body mass index within the normal range,of ASA physical status Ⅱ or Ⅲ (NYHA Ⅱ or Ⅲ),scheduled for elective cardiac valve replacement under CPB,were randomly divided into 2 groups (n =20 each) using a random number table:control group (group C) and APP group.In group APP,after induction of anesthesia,APP was performed,packed red blood cells and platelet-poor plasma were transfused back to the patient after termination of the previous cycle,and another cycle was started simultaneously.Platelet-rich plasma (PRP) was harvested and transfused back to the patient after termination of CPB and neutralization of heparin with protamine.Before induction of anesthesia (baseline),after APP and before heparinization,after neutralization of heparin with protamine and before back-transfusion of PRP,at the end of operation,and at 24 h after operation,venous blood samples were collected for determination of the expression of CD62p and PAC-1 in inactivated platelets and activated platelets by adenosine diphosphate (ADP).After APP and before heparinization,and after neutralization of heparin with protamine and before back-transfusion of PRP,the expression of CD62p and PAC-1 in inactivated platelets and ADP-activated platelets was detected in the whole blood and PRP in group APP.Results Compared with C group,the expression of CD62p and PAC-1 in inactivated platelets was down-regulated at the end of operation,and the expression of CD62p and PAC-1 in ADP-activated platelets was upregulated in APP group.The expression of CD62p and PAC-1 in inactivated platelets was down-regulated in PRP,and the expression of CD62p and PAC-1 in ADP-activated platelets was up-regulated in PRP as compared with those in the whole blood.Conclusion APP can not induce platelet activation,however,platelet back-transfusion can enhance platelet activation in the patients undergoing open heart surgery with CPB.

Objective Cryptococcosis is a potential life-threatening systemic mycosis with a heterogeneous susceptible popu-lation which is classified into three groups according to the current guidelines, including AIDS patients, organ transplantation recipients ( OTR) , and non-HIV-infected and non-transplant hosts ( NHNT) .This study aimed to discuss the influence of basic diseases on the clinical features and prognosis of NHNT cryptococcosis patients. Methods Using a retrospective cohort study design, we retrieved the clinical data about 73 NHNT cryptococcosis patients treated in Jinling Hospital.Based on the presence or absence of immunodefi-ciency or infection-increasing complications, we divided the patients into a basic disease group ( n=35) and a non-basic disease group ( n=38) and analyzed their clinical characteristics, chest radiographic features, laboratory results, and clinical outcomes. Results Compared with the non-basic disease group, the basic disease group showed a significantly higher incidence rate of disseminated disea-ses (62.9%vs 21.1%, P<0.01), more cases of patchy consolidation (47.4%vs 16.7%, P<0.05) and mixed lesion (31.6%vs 3.3%, P<0.05) in chest radiography, and a higher mortality (30.0%vs 5.3%, P=0.016). Conclusion Basic diseases have a great impact on the clinical features and prognosis of NHNT cryptococcosis.NHNT patients with basic diseases are susceptible to dis-seminated diseases with severer clinical symptoms and a higher mortality.

Objective Dendritic cell-associatedC-type lectin-1 ( Dectin-1) is one of the most important receptors in antifungal innate immune response.This study was to construct a recombinant adenovirus vector expressing themurine Dectin-1gene and acquire a high-concentration adenovirus by amplification and purification. Methods The PCR amplification product CLEC7A-pIRES2-EGFP was cloned into the intermediate vector pDONR221, and then recom-bined with the backbone vector pAD/CMV/V5-DEST to produce a re-combinant plasmid pAD-CLEC7A-pIRE2S -EGFP.The recombinant plasmid was linearized with Pac I and transfected into human embryon-ic kidney ( HEK293) cells to produce recombinant adenovirus pAD-CLEC7Ap-IRES 2-EGFP. The adenovirus was propagated in the HEK293 cells and purified by filtering through the cellulose acetate membrane and concentrating column.Fluorescence microscopy and re-al-time PCR were used to determine the expression of the Dectin-1 gene. Results PCR identification, enzyme digestion, and sequen-cing results manifested theDectin-1 gene in the vector, with the final adenovirus titer of 5×1011 IU/mL.Fluorescence microscopy revealed green fluorescence and real-time PCR assay confirmed that the expression of Dectin-1 was improved by 8677.25 times. Conclusion A relatively high-titer adenovirus expressing Dectin-1 was acquired,which may help to further study the high expression of Dectin-1 in anti-fungal innate immunity in vitro and in vivo.

Objective To explore the conditions and methods for cryopreservation and proliferation of mouse spermatogonial stem cells (SSCs). Methods SSCs were isolated from six-day-old Kunming mouse using two-step enzymatic digestion and Percoll discontinuous density gradient centrifugation. Cells were frozen with different freezing medium and cooling rate. After thaw, they were cultured in mimimum essential medium alpha (MEMα) supplemented with 10% fetal calf serum (FCS) and 100μg/L glial cell line-derived neurotrophic factor (GDNF). The survived and proliferating SSCs were examined by WST-8 colorimetric assay. Alkaline phosphatase andreverse transcription-polymerase chain reaction (RT-PCR) were performed to confirm if the cultured 96 hours germ cells were still stem cells. Results The best method to cryopreserve SSCs is using cryoprotector containing 10% dimethyl sulfoxide(DMSO), 10% FCS, 0.07mol/L sucrose and 1℃/min cooling rate, and the viability of cells in this method is more than 84%;Although the cell viability in non-programmed freezing method is less than that in the programmed freezing method, it is a simple and effective cropreservation method for mouse SSCs. What is more, the anchoring time of SSCs in this method is 8-12 hours after thaw, SSCs begin to proliferate 24 hours later, and rapid proliferation appears on the 48 hours, colonies are composed by 20-25 cells in 96 hours, when SSCs proliferated nearly 5 times.Conclusion The culture condition we used is suitable for proliferation of frozen-thawed SSCs.

Objective To understand the epidemiological characteristics of injury in college students in north jiangsu area ,a‐nalysis its related factors .Methods With the method of random cluster sampling ,1 857 students as the research object .Using self‐made questionnaire survey ,choose Chinese college students′ mental health scale (CCSM HS ) and Chinese perceived stress scale (CPSS) to investigate the mental health status of the research object .Results In 1 857 students ,639 students had more than once hurt ,harm the annual incidence of 34 .41% ;Incidence rate of injury was highest in junior college student(38 .94% ) ,lowest in gradu‐ate student(31 .75% ) .The highest constituent ratio of hurt types was fall/collision injury(34 .64% ) ,and lower extremity injuries was the main injured area .Education ,age ,ranking in the home ,parents′marital status ,students and their parents′physical condi‐tion ,the relationship with the students ,and the roommate to the occurrence of friction ,go out to work ,as well as the psychological stress test ,10 factors of college students′psychological scale ,10 factors oftotal pressure and tension factor were influencing factors of injury .Conclusion Damage and mental health problems is closely related .Safety education ,psychological education and interven‐tion should be carried out in order to effectively prevent and reduce the incidence of injury .