BACKGROUND:Both in vivo and vitro microenvironment can influence the proliferation and differentiation of neural stem cells (NSCs). In addition, cell culture solution plays variable roles in cell proliferation and differentiation.OBJECTIVE:To develop a convenient and rapid method to promote NSC primary culture by modifying traditional serum-free medium. DESIGN, TIME AND SETTING:Randomized controlled cell trial was performed at Institute of Brain Science, Qingdao University Medical School from November 2005 to September 2006.MATERIALS:Ten Wistar rat of gestation for 12-16 days; cell suspension fron brain tissue of embryonic rat. METHODS:Cell suspension were seeded into four 50-mL culture flasks with cell density of 1×106 mL-1, and divided into 2 groups with 2 flasks in each group. The control cells (flasks A and B) were cultured in serum-free medium containing DMEM/F12, B27 (2%), basic fibroblast growth factor (20 μg/L), and epidermal growth factor (20μg/L), and the experimental cells (flasks C and D) were firstly cultured with DMEM/F12 containing fetal bovine serum (FBS, 5%), following by the same serum-free medium 2 to 3 days later. MAIN OUTCOME MEASURES:The proliferation of NSCs was observed by inverted microscopy and immunocytochemistry.RESULTS:①Most of the cells in two culture conditions expressed nestin, the specific marker of NSCs, and were immunocytochemically positive. ②Neural stem cells cultured with FBS formed neurospheres 3-4 days earlier than those without FBS. ③There were no significant differences in cell number, but the neurospheres under the experimental condition were larger than those in control group. Some of the cells were neurone specific enolase-positive after serum-conditioned induction, and some were glial fibrillary acidic protein-positive, indicating NSCs had differentiated into neuron-like cells and neurogliocytes.CONCLUSION:FBS-conditioned pre-culture can accelerate the proliferation of neural stem cells.

Objective To investigate formalin-induced nociceptive behavior and c-fos expression of interbrain following stellate ganglionic block (SGB) in rabbits. Methods Catheters were inserted closely to right stellate ganglia in rabbits by operation.One week later,twenty-four rabbits were randomly divided into three groups, each group containing 8 animals. groups A,B and C received 0.5ml of normal saline,3% formalin and 3% formalin stimulation by intraplantar injection , respectively. 10 minutes before stimulation,0.5 ml of 0.25% bupivacaine was administered via the catheter in group B, while in groups A and C 0.5 ml of normal saline was applied. Nociceptive behavior was observed for 1 hour using weighted pain score. 2 hours after stimulation, whole interbrain was removed to immunohistochemically detect the expression of c-fos. Results Formalin-induced nociceptive behavior in phase 2 was relieved following SGB, c-fos expression level of hypothalamus in group B was significantly lower than that in group C(P0.05). Conclusion SGB could relieve formalin-induced nociceptive behavior, and downregulate formalin-induced c-fos expression of hypothalamus, which may be related to the mechanisms of SGB for the treatment of inflammatory pain.

Objective:To investigate the protective effect of dimethylsulfoxide(DMSO) on axon degeneration.(Methods: Cultured) rat superior cervical ganglia were treated with DMSO(100%,10 ?l) per well to disconnect axons from the cell bodies.SCGs in DMSO control group were treated with a mixture of DMSO(10 ?l) and medium (2 ml) per well;in positive control group were transfected with herpes simplex virus over-expressing Wld~S protein and the cell body was eliminated;and in blank control group were treated with 10 ?l PBS.The separated axons were fixed with 4% poly formaldehyde at 0,4,8,12 and 24 h after treatment with DMSO for immunostaining with specific antibody to microtubulin.Thus,the changes of axonal structure were investigated.The axonal protein was collected and the degeneration of neurofilament was detected by Western Blotting.Results: In DMSO disconnected group,the axonal morphology and structure showed no obvious change at 12 h post disconnection,but slight degeneration was observed at 24 h post disconnection.The degradation of microtubulin was obviously slowed down and their axonal structures maintained intact 12 h later.The neurofilament could be detected 12 h after disconnection.The above changes in disconnected group were similar to those in positive control group.No obvious protective effects on axonal degeneration were observed in blank and DMSO control groups.Conclusion: Local high concentration of DMSO can delay axonal degeneration,which indicates that DMSO can be used for adjuvant treatment of neurodegenerative diseases.

BACKGROUND: Ketamine is a kind of frequently used general venous anesthesia drug in clinic, and the medication in vein or epidural cavum has analgesic effect. It is N-methyl-D-aspartic acid (NMDA) receptor noncompetitive antagonist, which can inhibit toxic effect of excitatory amino acids.OBJECTIVE: To observe effect of ketamine on apoptosis of dorsal horn astrocytes of spinal cord of rats induced by NMDA receptor over activation and explore its possible mechanism of action.DESIGN: Randomized controlled observation.SETTING: Department of Anesthesiology, Taihe Hospital Affiliated to Yunyang Medical College.MATERIALS: The experiment was conducted at Cell Biology Laboratory,Institute of Basic Medical Sciences, Yunyang Medical College between September 2003 and January 2005. Neonatal Wistar rats of two or three days were provided by Animal Experimental Center of Wuhan University. METHODS: Primary astrocytes in dorsal horn of T11-L6 spinal cord of Wistar rats were purified and cultured. Astrocytes were used in the experiment when its purity coefficient reached 98% assessed by gial fibrillary acidic protein. The cultured cells in 24-well plates were divided randomly into 6 groups (9 portions in each group): ①50 μL Hanks liquor was added into the control group. ②Amount of 100μmol/L was added into the NMDA group. ③Amount of 1 mmol/L was added into the ketamine group. ④100μmol/L NMDA + 0.1 mmol/L ketamine group. ⑤100 μmol/L NMDA + 0.5 mmol/L ketamine group. ⑥100μmol/L NMDA + 1 mmol/L ke tamine group. 1 mmol/L ketamine was clinical antalgic dosage. Activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) were examined after 24-hour culture. Content of Bcl-2 protein and change of morphology were observed with immunocytochemistry. Apoptosis of astrocytes was measured with flow cytometry. MAIN OUTCOME MEASURES: ① Counterstain cell staining and changes of morphology of Bcl-2 protein with immunohistochemical method and hematoxylin-esoin staining (HE). ②Apoptosis of astrocytes was detected with flow cytometry. ③Content of MDA and activity of SOD.RESULTS: ①Mean absorbance (A) of Bcl-2 as expression of Bcl-2 protein measured semiquantitatively: It was lower in the 100μmoL/L NMDA group than the control group, which had significant difference [0.054±0.021,0.108±0.039, respectively, P＜0.01]. It was higher in the 100 μmol/L NMDA + 1 mmol/L ketamine group than the 100 μmol/L NMDA group,which had significant difference [0.148±0.045, 0.054±0.021, respectively,P ＜ 0.01]. ②Apoptosis of astrocytes detected with flow cytometry: It was higher in the 100μmol/L NMDA group than the control group, which had significant difference [(25.26±6.13)%, (5.66±2.24)%, respectively, P＜0.01].It was lower in the 100μmol/L NMDA + 1 mmol/L ketamine group than in the 100μmol/L NMDA group, which had significant difference[(24.41±4.82)%, (25.26±6.13)%, respectively, P＜0.01]. ③Content of MDA and activity of SOD: 100 μmol/L NMDA made the content of MDA in astrocytes obviously increase , while the activity of SOD markedly decrease. 1 mmol/L ketamine remarkably decreased the content of MDA, distinctly increased the activity of SOD. This effectiveness had evidently dosage-effect relationship in clinical antalgic dosage, which had obviously difference as compared with that of the NMDA group (P ＜ 0.01 ). The differences between the 1 mmol/L ketamine group and the control group as well as between the 100 μmol/L NMDA + 0.1 mmol/L ketamine group and the NMDA group had insignificant difference.CONCLUSION: NMDA receptor over activation can induce apoptosis of a great number of astrocytes in spinal dorsal horn of rats. Suitable ketamine dramatically inhibits apoptosis, and its mechanism can enhance the expressionof Bcl-2 protein of astrocytes, at the same time inhibit the production of free radical and reinforce the activity of SOD.

Objective: To investigate formalin-induced inflammatory nociceptive response and Fos expression of interbrain following stellate ganglion block (SGB) in rabbits. Methods: Catheters were inserted closely to right stellate ganglia in rabbits.One week later,twenty-four rabbits were randomly divided into three groups of 8 each:sham group,SGB group and control group,receiving 0.5ml of normal saline,3% formalin and 3% formalin stimulation by intraplantar injection into the right front paw, respectively. ten mins before stimulation,0.5ml of 0.25% bupivacaine was administered via the catheter in SGB group,while 0.5ml of normal saline in sham group and control group. Nociceptive response was observed for 60min using weighted pain score. Local inflammatory response was measured by histopathology, the expression of Fos of interbrain was measured with immunohistochemistry 2 hours after stimulation. Results: Formalin-induced nociceptive behavior in phase 2 and local inflammatory response were relieved following SGB; the average numbers of Fos-positive neurons of hypothalamus in SGB group was lower than that in control group( P 0.05). Conclusion: SGB suppresses formalin-induced Fos expression of hypothalamus.

Sixty-eight patients with pneumoconiosis combined with chronic obstructive pulmonary disease underwent large volume lavage in one lung under double cavity tracheal intubation and intravenousinhalant anesthesia. The vital signs of patients were recorded before, 10, 30min after and at the end of lavage. Results showed that the vital signs were stable during the lavage; and after the lavage all patients had relief significantly from the symptoms of dyspnea, polypnea and cough. Our results indicate that general anesthesia with bilateral lung ventilation are a safe and effective method in large volume whole lung lavage for treatment of pneumoconiosis patients combined with chronic obstructive pulmonary disease.

Objective To investigate the effect of stellate ganglion block ~SGB) on formalin-induced nociceptive behavior of rabbits, the expression of brain-derived neurotrophic factor ~BDNF) of dorsal root ganglia ~DRG) and the concentration of serum interleukin-8 ~IL-8) in rabbits. Methods Twenty-four rabbits were used in this study. Catheters were inserted closely to right stellate ganglia of the animals, which were randomly divided into control group ~A), SGB group ~B) and pain model group ~C). Group B and C were administered with an injection of 0.5ml of 3% formalin into the right front paw subcutaneously, group A 0.5ml saline by the same way. 0.5ml of (0.25)% bupivacaine was administered via the catheter in group B at 10 minutes before injection, while group A and C were given 0.5ml of saline at the same time. Nociceptive behavior was observed for 60 minutes after injection, and assessed by weighted pain score. Serum IL-8 was measured radioimmunologically at 10 minutes ~T_0) before injection, 10 ~T_1), 60 ~T_2) and 120 minutes ~T_3) after injection. The expression of BDNF in the right DRG of cervical 7, 8 and thoracic 1 was investigated immunohistochemically at 120 minutes after injection. Results Formalin-induced nociceptive behavior in phase Ⅱ was relieved following SGB. The concentration of serum IL-8 in groups B and C at T_2 and T_3 was significantly increased than that at T_0 and T_1~P

Objective To evaluate the role of mitochondrial ATP-sensitive potassium (mitoKATP) channel in mitigation of cerebral ischemia-reperfusion (I/R) injury by isoflurane preconditioning in rats and the relationship with c-Jun N-terminal kinase (JNK) signaling pathway.Methods Thirty-two male Sprague-Dawley rats,weighing 280-320 g,were randomly divided into 4 groups (n =8 each) using a random number table:sham operation group (group S),group I/R,isoflurane preconditioning group (group Ⅰ-pre),and 5-hydroxydecanoate (5-HD,a selective mitoKATP channel antagonist) group.Cerebral I/R was produced by modified 4-vessel technique described by Pulsinelli in anesthetized rats.In group Ⅰ-pre,the rats were exposed to 1.5％ isoflurane for 1 h everyday for 5 consecutive days before ischemia.In group 5-HD,5-HD 15 mg/kg was injected intraperitoneally at 30 min before ischemia and the other procedures were similar to those previously described in group Ⅰ-pre.Neurological behavior was evaluated at 24 h of reperfusion.The rats in each group were sacrificed at 72 h of reperfusion,and the brains were removed for determination of neuronal apoptosis (by TUNEL) and expression of caspase-3 and phosphor-JNK (p-JNK) protein (using Western blot) in hippocampal tissues.Apoptotic rate was calculated.Results Compared with group S,the number of grid cross was significantly decreased,hanging time was shortened,apoptotic rate was increased,and caspase-3 expression was up-regulated in I/R,Ⅰ-pre and 5-HD groups,the expression of p-JNK protein was up-regulated in IR and 5-HD groups,and no significant change was found in the expression of p-JNK protein in group Ⅰ-pre.Compare with group I/R,the number of grid cross was significantly increased,hanging time was prolonged,apoptotic rate was decreased,and the expression of caspase-3 and p-JNK protein was downregulated in group Ⅰ-pre,and no significant change was found in the parameters mentioned above in group 5-HD.Compared with group Ⅰ-pre,the number of grid cross was significantly decreased,hanging time was shortened,apoptotic rate was increased,and the expression of caspase-3 and p-JNK protein was up-regulated in group 5-HD.Conclusion The mitoKATP channel is involved in mitigation of cerebral I/R injury by isoflurane preconditioning through blocking the JNK signaling pathway in rats.

Objective To quantitate coronary tortuosity and provide favorable conditions for understanding the hemodynamic effects caused by coronary tortuosity.Methods We obtained all images from 72 patients who received coronary computed tomo-graphy scanning.After image post-processing,we extracted the medial axis of three main coronary vessel and analysed its coordi-nates on three dimensions.Then we calculated the tortuosity coefficient of coronary artery.Results Tortuosity coefficient was 6.66±7.54 in anterior descending,13.43±12.85 in left circumflex,and 1 7.61 ±7.67 in right coronary artery.We had proved its validity by the changes in morphology with simulated shapes.Conclusion We proposed a new method for quantitating coronary tor-tuosity,by computed tomography coronary imaging.The measurement results would not be affected by projection plane,vessel length or other artificial factors.

Objective To investigate the expression of SOX4 and C/EBPα mRNA in chronic myeloid leukemia (CML) and their clinical significances. Methods Bone marrow samples from 68 cases of CML including 57 newly diagnosed patients and 11 patients treated with imatinib were collected, and peripheral blood mononuclear cells from 30 healthy people were collected as healthy control. The expression of SOX4 and C/EBPαmRNA and protein levels were detected by RT-PCR and Western blot, respectively. The relations between the expression of SOX4 and C/EBPα and the influences of imatinib on SOX4 and C/EBPα were analyzed. Results The expression level of SOX4 mRNA was increased in newly diagnosed CML patients compared with that of normal control group (6.545 5±1.495 2 vs. 0.059 6±0.018 8, t=3.139, P=0.002 3), but the expression level of C/EBPαmRNA was significantly decreased (0.238 8±0.033 8 vs. 0.810 5±0.056 2, t=9.240, P<0.000 1). The expression levels of SOX4 and C/EBPαmRNA had no significant correlation with age, gender, white blood cell count (WBC) and bcr-abl of newly diagnosed patients (all P>0.05). The expression level of SOX4 mRNA in 5 patients treated with imatinib was decreased (0.120 6 ±0.044 9 vs. 0.557 9±0.144 8, t=2.885, P=0.020 4), and the expression level of C/EBPαmRNA was increased (0.330 3±0.042 4 vs. 0.150 5±0.046 5, t=2.855, P=0.021 3). The expression level of SOX4 mRNA in 6 patients who developed blast phase during the treatment of imatinib was increased (0.469 9±0.123 0 vs. 0.050 2±0.036 6, t=2.370, P=0.039 3), and the expression level of C/EBPα mRNA was decreased (0.197 9 ±0.064 7 vs. 0.378 7±0.042 9, t=2.327, P=0.042 3). The expression of SOX4 mRNA was negatively correlated with C/EBPα mRNA (t=-0.554 6, P=0.002 8). Conclusions In newly diagnosed CML, the expression level of SOX4 is increased, C/EBPα is decreased compared with that of healthy control, and both have negative correlation. In the patients in blast phase after imatinib treatment, SOX4 gene is up-regulated, and C/EBPα is down-regulated. C/EBPα-SOX4 axis may play a role in the occurrence and development of CML. SOX4 may be a new molecular target for the treatment of CML.