Objective To investigate the role of ca2+/calmodulin-dependent protein kinase II(CaMKII)in chronic inflammatory pain in mice.Methods Forty healthy male ICR mice weighing 20-25 g were randomly divided into 5 groups(n=8 each):group A control;group B chronic inflammation;group C KN92:group D KN93 30 nmol and group E KN93 45 nmo1.Chronic inflammatory pain was produced by injecting complete Freund's adjuvant(CFA) 20 ul into the dorsal surface of left hind paw in group B,C,D and E.In group A normal saline 20 ul was injected instead of CFA.On the 1st and 3rd day after CFA injection group D,E and Creceived intrathecal administration of specific CaMK Ⅱ inhibitor KN93 30 and 45 nmol and KN92 45 nmol(aninactive analog of KN93).in 5 ul respectively.Mechanical and thermal pain threshold to von Frey hair and radiantheat stimulation were determined at 30 min before CFA injection (To,baseline),30 min before(T1) and 30 min after IT.administration (T2) on the 1st day after CFA injection and at 30 min after IT administration(T3) on the 3rd day after CFA injection.The mice were immediately killed by C02 anesthesia after the last determination of pain threshold.The lumbar segment of the spinal cord Was then removed for determination of the expression of p-CaMK Ⅱ a by Western blotting.Results The thermal and mechanical pain threshold Was significantly decreasedat T1-3 in group B,C and D but only at T1 in group E as compared with the baseline values and group A.Thermal and mechanical pain threshold were significantly hisher at T2.3 in group E than in group B.p-CaMKⅡa expression in the spinal cord was significantly higher in group B,C and D than in group A but there was no significant difference in p-CaMKⅡa between group A and E.The p-CaMKII a expression in the spinal cord was significantyly lower in group E than in group B.Conclusion CaMK Ⅱ is involved in the development of chronic inflammatory pain.

Objective To establish an immortalized rat astrocyte strain(IAST) expressing enkephalin regulated by doxycycline(Dox) and observe its analysesic effect on rat chronic neuropathic pain. Methods Retrovirus infection method was employed to develop an immortalized rat astrocyte strain expression enkephalin regulated by doxycycline.hPPE gene expression level of IAST/Tet-On/hPPE strain was detected by Real time-PCR and radioimmunoassay.Its analgesic potential was investigated by mechanical paw withdrawal thresholds after these cells were implanted into the subarachnoid space of chronic constrictive injury(CCI) rats.The expression of Fos protein in the dorsal horn of spinal cord was determined by immunohistochemistry. Results An immortalized rat astrocyte strain secreting enkephalin under the control of doxycycline was established successfully.After transplantation of IAST/Tet-On/hPPE cell into the subarachnoid space of chronic constrictive injury(CCI) rats, the sensitivity of mechanical allodynia and the expression of Fos protein were significantly decreased(P

Objective: To compare effects of sufentanil with fentanyl when combined with propofol in intravenous anesthesia. Method: Forty ASA grade Ⅰ-Ⅱ patients, scheduled for abdominal operations, were randomly divided into two groups. Group sufentanil(Suf): combining sufentanil with propofol; group fentnyl (F): combining fentanyl with propofol. The items observed were perioperative hemodynamics, plasma epinephrine and norepinephine levels and analepsia time. Result: In group Suf, SP, DP, HR and RPP were decreased significantly (P0.05). In group F, the values above were also significantly decreased (P

ve To assess the protective effect of fentanyl on primary cultured myocardial cells against injury induced by anoxia-reoxygenation. Methods Ventricular myocardial cells enzymatically isolated and cultured in 1640 culture medium for 6 days were randomly divided into 5 groups: group A received no anoxia served as control; group B received 2 hours anoxia followed by half an hour reoxygenation; and group C, D and E received 10ng ? ml-1, 30 ng ? ml-1 and 50 ng? ml-1 fentanyl respectively before anoxia-reoxygenation. The cell viability, myocardial intracellular content of malondialdehyde (MDA) and the activities of lactic dehydrogenase (LDH) and creatine kinase(CK) were measured at the end of the experiment. Results Anoxia-reoxygenation caused dramatic decrease in cell viability, and increases in myocardial intracellular MDA content and the LDH and CK activities as compared with those in control group. Fentanyl 30 ng?ml-1 and 50 ng?ml-1 significantly attenuated the increases in LDH, CK activities and MDA content, and decrease in cell viability caused by anoxia-reoxygenation. Fentanyl 10ng?ml-1 did not produce any significant changes. Conclusions Fentanyl can produce protective effects on primary cultured cardiomyocytes against anoxia-reoxygenation injury.

Objective To investigate the effect of isovolumic hemodilution with 6% hydroxyethyl starch (HES) (200/0.5) on expression of intercellular adhesion molecule-1 (ICAM-1) after global cerebral ischemia-reperfusion in rats. Methods Eighty-four male Wistar rats weighing 230-280g were randomly divided into 3 groups: group Ⅱ sham operation (S , n = 20); group ischemia-reperfusion (Ⅱ , n = 32) and groupⅢ hemodilution with 6% HES (200/0.05) (H, n =32) . Group Ⅱ and group Ⅲ were further divided into 4 equal subgroups with 8 animals in each subgroup: 2h, 4h, 8h and 12h after beginning of reperfusion. Global cerebral ischemia was produced by permanent occlusion of bilateral vertebral arteries and cross-clamping of bilateral common carotid arteries for 10min and then clamping was released for reperfusion. In group Ⅲ acute normal volumic hemodilution was performed at 10 min after reperfusion was begun. 1ml/100g of blood was removed from artery and equal volume of 6% HES(200/0.5) was infused into the vein simultaneously. Hct was checked before and after hemodilution. The animals were decapitated at designed time and brain tissue was removed from ischemic area and frozen in liquid nitrogen. ICAM-1 expression was determined by using immunohistochemical technique. Results ICAM-1 expression significantly increased after 2h, 4h, 8h and 12h reperfusion in group Ⅱ and Ⅲ as compared with in group Ⅰ (P

Objective To study the biological character of rat astrocyte strain immortalized by simian virus 40 large T antigen gene(SV40Tag) and explore the feasibility of using it as cell vehicle for transgenic cellular analgesia.Methods Rat cerebral cortical astrocytes (AST) and immortalized rat astrocyte strain (IAST) were cultured in vitro. Morphology and growth features of AST and IAST were examined and compared after subculture, freezing and recovery. The ultrastructure of the cells was observed by transmission electron microscopy. The glial fibrillary acidic protein (GFAP) in these cells was detected by immuno-cytochemistry. The cell proliferation rate and cell cycle were determined by bromodeoxyuridine labelling and flow cytometry. AST and IAST were cultured in soft agar and inoculated in nude mice to investigate the tumorigenesis of IAST. Results LAST could be subcultured successively. The cells remained monolayer, anchorage dependent and the growth was attachment-inhibited. When AST was subcultured for only 10 passages, replicative senescense began. Subculture, freezing and recovery did not influence the shape and proliferation of IAST (94%?5%) , but decreased the vitality rate of AST (54%?4% ) (P

Objective To immortalize rat astrocytes which could be used as cell carriers for transgenic cellular analgesia. Methods Astrocytes were isolated from cerebral cortex of newborn SD rats by trypsin digestion and cultured according to the method of differential cell adhesiveness and transfected with plasmid pCMVSV40T/ PUR containing the simian virus 40 large tumor antigen (SV40Tag) gene. The positive colonies were isolated by puromycin selection and expanded by many passages. The integration and expression of large T antigen gene were detected and the immuno-reactivity of glial fibrillary acidic protein (GFAP) was determined by PCR, RT-PCR and immuno-cytochemistry. Results Rat astrocytes were successfully cultured in vitro and positively stained for the astrocytic marker GFAP. The positive colonies were isolated and subcultured for 50 passages. PCR and RT-PCR products were analyzed using 1.5% agarose gel electrophoresis. The size of amplifacation product of target gene was identical to that of the positive control (558 bp) . There was no PCR and RT-PCR product from non-transfected cells. DNA sequencing and BLAST showed that 558 bp nucleotides were identical to the SV40Tag gene of the Genbank (100%). The transfected cells were positively stained for the SV40Tag and GFAP. Conclusion Immortalized rat astrocyte strain with SV40 tag gene is constructed successfully.

Objective To construct a recombinant retroviral vector with human preproenkephalin gene regulated by tetracycline. Methods Human preproenkephalin gene was amplificated by polymerase chain reaction (PCR) and was cloned into retrovirus tetracycline responsive plasmid. Then this recombinant plasmid and regulatory plasmid pRev Tet-On were transferred into packaging cell PT67 respectively. The transfected PT67 / Tet-On and PT 67 / TREhPPE cells were selected by the corresponding antibiotics and identified by RT-PCR. The selected cells were enriched and virus liter was assayed using NIH3T3. Results The restriction endonuclease digestion, PCR analysis and DNA sequencing confirmed that the recombinant RevTRE/hPPE vector was constructed successfully. The virus liter of PT 67/TREhPPE was 3.2 ? 103 CFU?ml-1 and the virus titer of PT67/Tet-On was 2.6?105 CFU?ml-1.Conclusion A recombinant retroviral vector with human preproenkephalin gene regulated by trtracycline and stable virus producing lines have been successfully constructed, providing a good basis for further research on regulated cell therapy of chronic pain.

Objective To establish the surgical landmarks of the endoscopic endonasal approach to the ventral region of middle-lower part of clivus and provide anatomic basis. Methods Twenty 10% formalin-fixed intact adult head specimens were used to dissect and observe the anatomic feature of this access in order to establish the surgical landmarks of the approach, and some relative anatomic data were measured. Five fresh and intact head specimens injected with colored latex were used, and completely analogical operation via endoscopic endonasal approach to the middle-lower part of clivus was performed in all cases. Results Anatomic landmarks of the approach included middle turbinate, choana narium, eustachian tube ostium, nasopharynx mucosa, longus capitis and longus colli, pharyngeal tubercle, and basi-on. To expose the ventral region of middle-lower part of clivus completely, the shortest distance was ( 89.60 ± 2. 52) mm. The ranges of stripping the inferior wall of sphenoid sinus and the lower clivus were bounded by pterygoid canal and foramen lacerum, and the distances from the median line were (9. 37 ± 0.59) mm and (10. 75 ± 0. 63 ) mm, respectively. Conclusions The structures of the ventral middle-lower part of clivus can be revealed sufficiently via an endoscopic endonasal approach.

BACKGROUND: Recently, lithium was reported shown neuroprotective effect against apoptosis induced by a variety of insults in vitro and in vitro,but the precise mechanisms underlying its neuroprotective effect remain unknown.OBJECTIVE: To observe the effect of lithium chloride on neuronal apoptosis and the expression of P53 or nuclear factor kappa B (NF-κB) protein in the CA1 region of the hippocampus after global ischemia in gerbils.DESIGN: A randomized controlled experimental research.SETTING: Department of Anatomy of Nanjing Medical University.MATERIALS: Fifty-four healthy male gerbils weighing 50-70 g, clearing grade, were purchased from Experimental Animal Center of Zhejiang Province.METHODS: Totally 54 gerbils were randomly divided into three groups namely: sham-operation group (SH group), ischemia-reperfusion group (IR group) and lithium chloride group (LI group), with 18 in each group. SH group, IR group and LI group were further divided into 3 subgroups respectively (SH1d, SH3d, SH7d; IR1d, IR3d, IR7d; LI1d, LI3d, LI7d), according to the time of reperfusion, with 6 gerbils in each. Gerbils in LI group were injected intraperitoneally with lithium chloride 3 mEq /kg, once a day for 7consecutive days before operation. Normal saline was used instead of lithium in SH group and IR group as vehicle control. Forebrain ischemia was induced at 24 hours after the last injection of lithium chloride. After gerbils being anesthetized, the bilateral common carotid arteries were blocked with micro aneurysm clips for 5 minutes, and the micro aneurysm clips were removed and the cerebral blood flow restored. Sham-operation animals were underwent the same operation except occlusion of bilateral common carotid arteries. Gerbils in each group were killed at every time points.4 μm coronal sections at 1.7-4.0 mm visual cross were cut at the level of the dorsal hippocampus. The apoptosis cells were assayed with in situ Cell Death Detection Kit, and assay of positive cell in cell apoptosis, P53 and positive NF-κB was performed with immunohistochemistry staining. The total number of TUNEL positive cells, P53 or NF-κB positive cells per image (area of 1 mm2) was counted.MAIN OUTCOME MEASURES: Neuronal apoptosis and expression of P53 or NF-κB protein in the CA1 region after cerebral ischemic reperfusion.apoptosis cell in cerebral hippocampus CA1 region: No TUNEL positive cells were detected in SH group, a large majority of TUNEL positive cells were detected in the CA1 region in IR group on the 3rd day after reperfusion [(552.0±145.5, 142.4±103.5) pcs/mm2, t= 5.623, P ＜ 0.01], and TUNEL positive cells declined on the 7th day after reperfusion. The numbers of TUNEL positive cells in the CA1 region of LI3d, LI7d group were significantly lower than those of IR3d, IR7d group [(408.0±119.8, 156.0±108.2) pcs/mm2,CA1 region: In IR group, the expression of P53 protein was increased on the 1st, 3rd and 7th day after reperfusion compared with that in SH group and cerebral hippocampus CA1 region: No NF-κB protein was expressed in SH group. In IR group, the expression of NF-κB protein was increased on the 1st day after reperfusion (78.5±25.2)/mm2, significantly increased on the 3rd day after reperfusion (176.5±35.5)/mm2 and on the 7th day after reperfusion, the expression of NF-κB protein disappeared. There were no significant statistical difference between LI group and IR group on the 1st day after reperfusion. The expression of NF-κB protein in LI group was significantly lower than that in IR group on the 3rd day after reperfusion [(64.5±30.8)/mm2,t=5.824, P ＜ 0.01].CONCLUSION: Lithium chloride can significantly suppress neuronal apoptosis after global ischemia in gerbils. The down-regulation of expression of P53 or NF-κB protein is one of the mechanisms of the neuroprotective effect by lithium chloride.