Objective To investigate the relationship between autophagy in spinal dorsal horn and development of morphine tolerance in rats.Methods Twenty-four healthy male Sprague-Dawley rats,in which intrathecal (IT) catheters were successfully placed,were randomly divided into 3 groups (n =8 each):control group (group C),morphine tolerance group (group M) and morphine + rapamycin as a reinforcing agent for autophagy group (group MR).Morphine tolerance was induced with IT morphine 20 μg twice a day for 7 consecutive days.While the equal volume of normal saline was given in group C.In addition,rapamycin 2.3μg was injected intrathecally at the second injection of morphine on 3rd day lasting for 3 consecutive days in group MR.Mechanical withdrawal threshold (MWT) to yon Frey filament stimulation was measured before IT injection and 30 min after the second IT injection on 1st,3rd,5th and 7th days.The rats were sacrificed 1 h after the last MWT measurement and the L4-6 segment of the spinal cord was removed for determination of the total mammalian target of rapamycin (mTOR) and phosphorylated mTOR(p-mTOR) and autophagy marker protein LC3 Ⅱ expression in spinal dorsal horn by Western blot.The percentage of p-mTOR expression in total mTOR expression was considered as reflection of the activity.Results MWT was gradually decreased with the prolongation of time of IT injection (P ＜ 0.05).Compared with group C,MWT was significantly increased during IT injection,mTOR activity was decreased and LC3 Ⅱ expression was up-regulated in groups M and MR (P ＜ 0.05).Compared with group M,MWT was significandy increased on 3rd,5th and 7th days after IT injection,mTOR activity was decreased and LC3 Ⅱ expression was up-regulated in group MR (P ＜ 0.05).Conclusion Increased autophagy in spinal dorsal horn is the regulatory mechanism of the body during the development of morphine tolerance in rats and can delay the development of morphine tolerance.

Objective To review the treatment results of intracytoplasmic injection(ICSI) of epididymal or testicular sperm obtained from 38 obstructive azoospermic patients.Methods Sperm was retrieved by percutaneous epididymal sperm aspiration(PESA) or testicular sperm extraction(TESE).Intracytoplasmic injection was performed.The rates of fertilization and clinical pregnancy were evaluated.Control group was set up in which intracytoplasmic injection was performed using sperm of ejaculation.Results Forty-one treatment cycles were performed in the 38 obstructive azoospermc patients.The rates of fertilization and clinical pregnancy were 73.3% and 53.6%.Thirty-three treatment cycles were done in the 31 ejaculatory ones.The rates of fertilization and clinical pregnancy were 75.1% and 48.4%.No significant difference was seen between the two groups.In the obstructive azoospermia group,22 clinical pregnancies were achieved including 13 live deliveries and 3 ongoing pregnancies and 6 miscarriages.In the ejaculatory group,16 clinical pregnancies were achieved including 10 live deliveries and 5 ongoing pregnancies and 1 miscarriages.Conclusions ICSI with PESA or TESE is an effective method for treatment of obstructive azoospermic patients.

Objective To explore the application value of preoperative methylene blue staining in locating for the operation of intraspi-nal tumors. Methods The clinical data of patients with intraspinal tumors from September 2010 to September 2012 in our hospital were ret-rospectively analyzed. The patients were divided into tag group and control group according to whether stained by methylene blue or not. The operation time( min) ,intraoperative hemorrhage,the rate of total resection of tumor,spinal instability rate,tumor recurrence rate,and reopera-tion rate of two groups were compared. Results The operation time of tag group was significantly shorter than that of the control group. The amount of intraoperative bleeding was significantly less than that of in control group, the differences were statistically significant(P<0. 05). The total resection rate of tumor was significantly higher than that in control group,the differences were statistically significant(P<0. 05). The spinal instability rate,tumor recurrence rate and operation rate of patients within 1 year in two groups were not significant. Conclusion The methylene blue method is simple and convenient,and provides favorable conditions for the operation,which reduces the operation time and intraoperative hemorrhage,increases the rate of complete tumor resection. There was no difference in recurrence rate,operation rate and the stability of the spine within 1 year compared to traditional method.

Timely reporting, effective analyses and rapid distribution of surveillance data can assist in detecting the aberration of disease occurrence and further facilitate a timely response. In China, a new nationwide web-based automated system for outbreak detection and rapid response was developed in 2008. The China Infectious Disease Automated-alert and Response System (CIDARS) was developed by the Chinese Center for Disease Control and Prevention based on the surveillance data from the existing electronic National Notifiable Infectious Diseases Reporting Information System (NIDRIS) started in 2004. NIDRIS greatly improved the timeliness and completeness of data reporting with real time reporting information via the Internet. CIDARS further facilitates the data analysis, aberration detection, signal dissemination, signal response and information communication needed by public health departments across the country. In CIDARS, three aberration detection methods are used to detect the unusual occurrence of 28 notifiable infectious diseases at the county level and to transmit that information either in real-time or on a daily basis. The Internet, computers and mobile phones are used to accomplish rapid signal generation and dissemination, timely reporting and reviewing of the signal response results. CIDARS has been used nationwide since 2008; all Centers for Disease Control and Prevention (CDC) in China at the county, prefecture, provincial and national levels are involved in the system. It assists with early outbreak detection at the local level and prompts reporting of unusual disease occurrences or potential outbreaks to CDCs throughout the country.

OBJECTIVETo study the clinicopathologic features and significance of aberrant CD56 expression in diffuse large B-cell lymphoma (DLBCL).

METHODSThe clinical and pathologic profiles of 10 cases of DLBCL with aberrant expression of CD56 were investigated. Immunohistochemical staining, in-situ hybridization for Epstein-Barr virus encoded RNA and gene rearrangement for IgH and Igκ were carried out.

RESULTSThere were 6 male and 4 female patients. The medium age of patients was 46 years. All of them presented with extranodal lymphoma involvement, with gastrointestinal tract being the commonest site (5/10). Histologic examination showed that most of the atypical lymphoid cells were centroblast-like and demonstrated a diffuse growth pattern. Apoptosis and necrosis were identified in some cases. Immunohistochemical study showed that the tumor cells were positive for CD20 or CD79α and aberrantly expressed CD56. Five cases had the GCB phenotype while the remaining cases had the non-GCB phenotype, according to Hans classification. Bcl-6 was positive in most cases (9/10). All cases showed a high proliferation index by Ki-67. The tumor cells were negative for CD3ε, CD138 and granzyme B. In-situ hybridization for Epstein-Barr virus encoded RNA was performed in 7 cases and none of them showed positive signals. IgH gene rearranged bands were detected in 4 cases (4/6) and Igκ was detected in 3 cases (3/6). Follow-up data were available in 8 patients. Two patients died of disease progression within 5 to 13 months after diagnosis and the other 6 patients were alive 8 to 60 months after therapy.

CONCLUSIONSDLBCL with aberrant expression of CD56 is rare. Most of them present with extranodal involvement, show high frequency of bcl-6 expression and high proliferation index. The patients often have good response to chemotherapy.

Antigens, CD20 ; metabolism ; Apoptosis ; CD56 Antigen ; metabolism ; CD79 Antigens ; metabolism ; Disease Progression ; Female ; Gene Rearrangement ; Granzymes ; metabolism ; Herpesvirus 4, Human ; genetics ; Humans ; Immunophenotyping ; In Situ Hybridization ; Lymphoma, Large B-Cell, Diffuse ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Necrosis ; Phenotype ; Proto-Oncogene Proteins c-bcl-6 ; metabolism ; RNA, Viral ; analysis

Objective To establish virtual three-dimensional models of interventional devices and individualized surgical area structure in transjugular intrahepatic portosystemic shunt(TIPS)treatment,and to explore the value of virtual models for preoperative simulation in TIPS treatment.Methods Thin slice scan data of 8 patients with supine upper abdomen were obtained,three dimensional structures of bone,liver,portal vein,inferior vena cava and hepatic vein in CT scan area were reconstructed in Mimics software.According to the size of interventional instruments,a virtual RUPS-100 puncture kit and an VIATORR stent were established in 3D MAX software. Computer simulations were performed to evaluate the route from the hepatic vein puncture portal vein using the RUPS-100 puncture kit and VIATORR stent release position.Results ①The establishment of individual three-dimensional model of patients was helpful for doctors to understand the spatial relation of hepatic vein and portal vein.②Through simulation,the puncture parameters of multi angle and multi position are obtained,which was helpful for the doctor to adjust the puncture direction according to the puncture point.③The position of the bare segment of the VIATORR stent in the portal vein was obtained by simulation,which was helpful for evaluating the length of the stent.④The preoperative simulation results included the simulation parameters for each patient puncture into left portal vein,right portal vein and portal vein bifurcation.In the actual operation,we punctured into the portal vein bifurcation in 4 cases,into the right branch in 2 cases and into the left in 2 cases.⑤Preoperative simulations were performed using 8 mm×6 cm×2 cm size VIATORR stent.Howere,the actual operation of the first case was lack of experience,and the stent position was lower,then we released a bare stent at the proximal end of the VIATORR stent.The rest of the cases were the same as the simulation results.Conclusion According to the three-dimensional model of the individual structure of the patient,the preoperative simulations have high fidelity.The simulation results of the parameters of the puncture and the release position of the stent could guide the actual operation more accurately.It is of practical value to improve the success rate of operation and to train residents.

Objective:To investigate the changes of autonomic nervous active substances in myocardium of rats with acute high-level spinal cord injury.Methods:Twenty-four clean-level healthy adult male SD rats weighting 250-300 g for 8-10 weeks old were divided into control group ( n=6) and spinal cord injury group ( n=18) according to the random number table. The spinal cord injury group was subdivided at 4, 12 and 24 hours, with 6 rats at each time point. The high-level spinal cord injury model was established by the modified Allen′s weight-drop method, and the spinal cord was only exposed in control group. The postoperative performance and BBB score for limb movement were observed in each group. The myocardium of each group was resected and used to observe ultrastructure of myocardial cells under transmission electron microscope and detect protein and mRNA levels of tyrosine hydroxylase (TH), noradrenaline transporter (NET), acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) by Western blot and RT-PCR analysis. Results:Rats of control group showed normal limb motion after operation without significant change from the preoperation level, and mean BBB score was 21 points. Rats of spinal cord injury group showed significantly reduced activities and feeding, with flaccid paralysis of both lower limbs as well as no spontaneous excretion, and showed BBB score of 0 point at 4 hours and 12 hours after injury, which was increased slightly at 24 hours after injury, with the highest score for 1 point. The ultrastructure of myocardial cells showed no obvious abnormalities in control group, while different degrees of changes in spinal cord injury group. Compared with control group, Western blot analysis showed that protein levels of TH and NET were decreased, while AChE and ChAT were increased in spinal cord injury group ( P<0.05 or 0.01). Compared with control group, RT-PCR analysis showed that mRNA levels of TH and NET were decreased, while AChE and ChAT were increased in spinal cord injury group ( P<0.05 or 0.01). mRNA levels of TH and NET in spinal cord injury group at 24 hours after injury were significantly different from those at 4 hours and 12 hours after injury (all P<0.05). mRNA levels of ChAT in spinal cord injury group were statistically significant at 12 hours and 24 hours after injury from those at 4 hours after injury, with significant difference at 12 hours and 24 hours after injury (all P<0.05). Conclusion:Sympathetic nerve active substances TH and NET are down-regulated but vagal nerve active substances AChE and ChAT up-regulated in myocardium of rats with acute high-level spinal cord injury, which may be related to the relative excitation of the parasympathetic nerve blocking the sympathetic innervation of the higher center to the heart following high-level spinal cord injury.

Objective An HPLC-MS/MS method was established for the simultaneous determination of 7 components in Qingkailing Oral Liquid.Methods The assay was performed on a Waters ACQUITY UPLC BEH C18 column(2.1 mm×10 mm,1.7 μm)and the sample was eluted with a gradient mobile phase containing 10 mmol·L-1 of ammonium acetate and 0.1%of formic acid in water(A)-methanol(B).The mass spectrometry was carried out by electrospray ionization(ESI)with positive/negative ions in multiple reaction monitoring(MRM)mode for quantitative analysis.Results The linear ranges of adenine,chlorogenic acid,caffeic acid,geniposide,baicalin,hyodeoxycholic acid and cholic acid were 0.100 4-3.213,0.784 5-8.982,0.998-3.194,0.622 5-19.92,25.05-300.6,2.513-30.15 and 7.775-93.30 μg·mL-1(r≥0.999 0).The average recoveries(n=6)were 100.9%,98.74%,101.2%,100.2%,100.8%,99.97%and 98.94%with RSD of 1.58%,0.59%,1.78%,1.25%,0.65%,1.69%and 1.07%.The contents of the above mentioned 7 components in 15 tested samples were in the ranges of 0.12-0.18,0.19-0.24,0.06-0.09,0.34-0.37,4.54-4.85,0.49-0.67 and 1.82-2.19 mg·mL-1.The contents of 7 components in tested sample from different manufacturers were closed.Conclusion The method has shown good sensitivity,accuracy,and repeatability.The study can provide reference and data support for the quality control and subsequent research of Qingkailing Oral Liquid.

Objective:To investigate the effects of astragaloside IV (AS-IV) on acute myocardial injury in rats with high-level spinal cord injury (SCI).Methods:Twenty-four healthy male SD rats, aged 8-10 weeks with a body weight of 250-300 g, were randomly divided into 4 groups using a random number table method: sham operation group, high-level SCI group (SCI group), high-level SCI+AS-IV group (SCI+AS-IV group) and high-level SCI+AS-IV+silent information regulator 1 (SIRT1) inhibitor EX527 group (SCI+AS-IV+EX527 group), with 6 rats in each group. The SCI model was established using the modified Allen method and the sham operation group underwent the spinal cord exposure only. In the SCI+AS-IV group, 40 mg/kg of AS-IV was injected intraperitoneally immediately after injury. SCI+AS-IV+EX527 group received an intraperitoneal injection of 5 mg/kg EX527 at one hour before injury and another injection of 40 mg/kg AS-IV in the same way immediately after injury. The sham operation group and the SCI group received an equal volume of saline via intraperitoneal injection. Immediately after awakening from injury, the hind limb motor function of the rats in each group was observed, recorded and then evaluated using the BBB method. At 24 hours after injury, the ultrastructure of the cardiomyocytes was examined under a transmission electron microscope; the levels of serum cardiac troponin I (cTnI), myocardial tissue inflammatory factors interleukin (IL)-18 and IL-1β were quantified by the ELISA method; the level of reactive oxygen species (ROS) of the myocardial tissue was assessed utilizing the dihydroethidium (DHE) assay; biochemical analyses were employed to determine the superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentrations; mRNA and protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), cysteinyl aspartate specific proteinase-1 (caspase-1), gasdermin D (GSDMD), SIRT1 and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) were examined using RT-PCR and Western blot; cardiomyocyte pyroptosis rate was evaluated by caspase-1 and TUNEL double-labeled fluorescence staining.Results:Immediately after awakening from injury, the sham operation group exhibited normal hind limb activity, with BBB scores of 21(21, 21)points, while the remaining groups displayed flaccid paralysis in both hind limbs, accompanied by the cessation of spontaneous excretion, with BBB scores of 0(0, 0)points. At 24 hours after injury, transmission electron microscopy did not reveal any significant abnormalities in the ultrastructure of the myocardiomyocytes in the sham operation group, while changes of varying degrees were observed in the SCI group. The ELISA results indicated that at 24 hours after injury, the serum cTnI level in the SCI group was (1 435.3±148.1)pg/ml, higher than (619.6±95.4)pg/ml in the sham operation group ( P<0.01); the cTnI level was (1 154.0±80.0)pg/ml in the SCI+AS-IV group, lower than that in the SCI group ( P<0.01); the cTnI level was (1 321.8±50.2)pg/ml in the SCI+AS-IV+EX527 group, higher than that in the SCI+AS-IV group ( P<0.05). The levels of IL-18 and IL-1β in the myocardial tissue in the SCI group were (493.0±145.0)pg/ml and (936.7±93.2)pg/ml, higher than (131.1±62.5)pg/ml and (281.7±83.6)pg/ml in the sham operation group ( P<0.01); the levels of IL-18 and IL-1β in the SCI+AS-IV group were (182.4±45.6)pg/ml and (573.4±99.5)pg/ml, lower than those in the SCI group ( P<0.01); the levels of IL-18 and IL-1β in the SCI+AS-IV+EX527 group were (337.4±72.0)pg/ml and (742.6±82.7)pg/ml, higher than those in the SCI+AS-IV group ( P<0.05), yet lower than those in the SCI group ( P<0.01). At 24 hours after injury, DHE and biochemical assays showed that the levels of ROS and MDA in the myocardial tissue in the SCI group were (65±6)% and (1.97±0.27)nmol/mg, higher than (19±10)% and (1.03±0.16)nmol/mg in the sham operation group ( P<0.01); the ROS and MDA levels in the SCI+AS-IV group were (37±10)% and (1.39±0.11)nmol/mg, lower than those in the SCI group ( P<0.01); the ROS and MDA levels in the SCI+AS-IV+EX527 group were (52±7)% and (1.70±0.14)nmol/mg, higher than those in the SCI+AS-IV group ( P<0.05). The SOD level in the myocardial tissue of the SCI group was (658.48±77.56)U/mg, lower than (1 059.55±71.91)U/mg in the sham operation group ( P<0.01); the SOD level in the SCI+AS-IV group was (901.74±32.30)U/mg, higher than that in the SCI group ( P<0.01); the SOD level in the myocardial tissue in the SCI+AS-IV+EX527 group was (799.86±26.70)U/mg, lower than that in the SCI+AS-IV group ( P<0.05). At 24 hours after injury, RT-PCR showed that the mRNA expression levels of NLRP3, caspase-1 and GSDMD in the myocardial tissue of the SCI group were 2.07±0.25, 2.46±0.28 and 1.82±0.12 respectively, which were higher than 1.10±0.13, 0.95±0.17 and 1.03±0.08 in the sham operation group ( P<0.01); the mRNA expression levels of NLRP3, caspase-1 and GSDMD in the SCI+AS-IV group were 1.47±0.24, 1.51±0.16 and 1.42±0.13 respectively, which were lower than those in the SCI group ( P<0.01); the mRNA expression levels of NLRP3, caspase-1 and GSDMD in the SCI+AS-IV+EX527 group were 1.93±0.28, 1.97±0.31 and 1.65±0.16 respectively, which were higher than those in the SCI+AS-IV group, yet lower than those in the SCI group ( P<0.05). The mRNA expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI group were 0.41±0.09 and 0.56±0.07, lower than 1.20±0.14 and 1.29±0.20 in the sham operation group ( P<0.01); the mRNA expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI+AS-IV group were 0.78±0.08 and 1.01±0.19, higher than those of the SCI group ( P<0.01); the mRNA expression levels of SIRT1 and PGC-1α in the myocardial tissue of the SCI+AS-IV+EX527 group were 0.53±0.12 and 0.72±0.22, lower than those of the SCI+AS-IV group ( P<0.05). At 24 hours after injury, the western blot analysis showed that the protein expression levels of NLRP3, caspase-1 and GSDMD in the myocardial tissue in the SCI group were 1.00±0.20, 0.60±0.19 and 0.77±0.15 respectively, which were higher than 0.27±0.09, 0.18±0.10 and 0.28±0.08 in the sham operation group ( P<0.01); the protein expression levels of NLRP3, caspase-1 and GSDMD in the SCI+AS-IV group were 0.59±0.10, 0.25±0.11 and 0.33±0.11 respectively, lower than those in the SCI group ( P<0.01); the protein expression levels of NLRP3, caspase-1 and GSDMD in the myocardial tissue in the SCI+AS-IV+EX527 group were 0.85±0.15, 0.54±0.12 and 0.55±0.13 respectively, higher than those in the SCI+AS-IV group ( P<0.05). The protein expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI group were 0.44±0.16 and 0.28±0.10, lower than 0.93±0.22 and 0.75±0.16 in the sham operation group ( P<0.01); the protein expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI+AS-IV group were 0.78±0.19 and 0.55±0.12, higher than those in the SCI group ( P<0.01); the protein expression levels of SIRT1 and PGC-1α in the myocardial tissue in the SCI+AS-IV+EX527 group were 0.46±0.16 and 0.35±0.07, lower than those in the SCI+AS-IV group ( P<0.05). At 24 hours after injury, caspase-1 and TUNEL double-labeled fluorescence staining showed that the cardiomyocyte pyroptosis rate in the SCI group was (34.5±6.7)%, higher than (5.3±2.9)% in the sham operation group ( P<0.01); the cardiomyocyte pyroptosis rate in the SCI+AS-IV group was (13.4±3.0)%, lower than that in the SCI group ( P<0.01); the cardiomyocyte pyroptosis rate in the SCI+AS-IV+EX527 group was (22.5±5.9)%, higher than that in the SCI+AS-IV group ( P<0.01), yet lower than that in the SCI group ( P<0.01). Conclusions:AS-IV can significantly reduce acute myocardial injury in rats with high-level SCI. Its mechanism may involve activating the myocardial SIRT1/PGC-1α signaling pathway, protecting the mitochondria, enhancing the ability to resist oxidative stress, and effectively inhibiting the NLRP3 inflammasome-mediated pyroptosis pathway.

ObjectiveTo investigate the effects of Huashi Runzao prescription （HRP） on the histopathological injury and function of submandibular gland in naive non-obese diabetic （NOD/Ltj） mouse model of Sjögren's syndrome （SS） and its regulatory effect on aquaporin 5 （AQP5） expression in submandibular gland cells. MethodThe SS model was induced in NOD/Ltj mice. The NOD/Ltj female mice aged nine weeks were selected and randomly assigned into model group，HRP group （7.15 g·kg^-1·d^-1），and hydroxychloroquine （HCQ） group （1.30 g·kg^-1·d^-1）， and female BALB/c mice in the same age were selected and assigned into the normal group， with six mice in each group. Drug intervention lasted eight weeks. The water consumption and salivary flow rate （SFR） of each group were recorded. The pathological staining results of the submandibular gland of mice in each group were observed and scored. AQP5 expression was determined by immunohistochemistry （IHC） and Western blot. ResultCompared with the normal group， the model group showed increased water consumption （P<0.05） and reduced SFR （P<0.05）. Compared with the model group， the HRP group showed decreased water consumption （P<0.05） and increased SFR （P<0.05）， and the HCQ group showed increased SFR （P<0.05）. In terms of histopathological results of the submandibular gland，compared with the normal group，the model group showed increased pathological score， number of lymphocyte infiltration foci，and percentage of lymphatic infiltration area （P<0.05）. Compared with the model group， the HRP group showed reduced pathological scores and number of lymphocyte infiltration foci （P<0.05）， and the HRP group and the HCQ group showed reduced percentage of lymphatic infiltration area（P<0.05）. The results of IHC and Western blot showed that compared with the normal group，the model group showed down-regulated expression level of AQP5 protein （P<0.05）， and compared with the model group and the HCQ group，the HRP group showed up-regulated expression level of AQP5 protein （P<0.05）. ConclusionHRP can improve the secretion function of submandibular gland acinous cells and glandular structure injury in SS model mice， and its mechanism may be related to the up-regulation of AQP5 protein expression level in submandibular gland cells.