BACKGROUND: Recently, several scientists have found that differentiation of neural stem cells (NSCs) towards dopaminergic neurons may be increased in vitro by combination of some special cytokines. They have also found that dopaminergic neurons differentiated from NSCs can be used for the treatment of Parkinsn's disease. To improve the therapeutic effects of/n vitro transplantation, we should further study the biologic characteristics of NSCs at the induction and differentiation.OBJECTIVE: To explore the differentiation of NSCs which were incubated in differentiation solution for different time towards dopaminergic neurons in vitro.DESIGN: Single sample observation.SETTING: Department of Neurosurgery, First Affiliated Hospital of Sun Yat-sen University.MATERIALS: This study was performed at the Department of Ncurosurgery, First Affiliated Hospital of Sun Yat-sen University from May to October 2007. Six healthy Sprague Dawley (SD) rats, gestational age 14 days, of clean grade, weighing 350-400 g,were provided by the Laboratory Animal Center, Sun Yat-sen University[permission No. SCXK (yue)2007-0034]. The protocol was performed in accordance with ethical guidelines for the use and care of animals.METHODS: NSCs derived from rat embryonic mesencephalon were cultured in serum-free culture medium containing epidermal growth factors and basic fibroblast growth factors. After passage, the NSCs were induced to differentiate towards dopaminergic neurons in the differentiation medium supplemented with interleukin 1o, interleukinl 1, human leukaemia inhibitory factors, and glial cell line-derived neurotrophic factors. The percentage of tyrosine hydroxylase positive neurons in differentiated ceils was detected with flow cytometer when NSCs were cultured in differentiation solution for 2, 4, 6, 8 and 10 days, respectively.MAIN OUTCOME MEASURES: Cellular morphological alteration of rat NSCs after differentiation. The percentage of tyrosine hydroxylase positive neurons in differentiated cells derived from NSCs.RESULTS: In differentiation medium, NSC spheres attached the bottom of plates and began to collapse. Cells inside the spheres grew out gradually and became irregular in shape. Six days later, most of the cells had I or 2 long processes and a few short processes. The percentage of tyrosine hydroxylase positive neurons in differentiated cells was respectively (3.2_+_0.9)%, (6.8 +1.6)%, (16.7-+2.6)%, (14.8_+1.8)% and (12.2_+2.5)% after culture for 2, 4, 6, 8 and 10 days, with significant differences (F =26.449, P ＜ 0.05).CONCLUSION: Induction time influences the differentiation of NSCs towards dopaminergic neurons in vitro. The percentage of dopaminergic neurons is the highest in differentiated cells derived from NSCs which are cultured in differentiation solution for 6 days.

BACKGROUND: Differentiation inducing factors and gestational age influence the differentiation potential of embryonic neuralstem cells.OBJECTIVE: This study was designed to observe the differentiation potential of rat mesencephalic neural stem cells at differentgestational ages towards dopaminergic neurons.DESIGN: A randomized controlled observation.SETTING: Department of Neurosurgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou City, GuangdongProvince, China.MATERIALS: This study was performed at the Laboratory of the First Affiliated Hospital of Sun Yat-sen University between Marchand September 2007. Thirty adult gestational SD rats, weighing 350 400 g, were provided by the Laboratory Animal Center of SunYat-sen University (Permission No. 2007-0034). The protocol was performed in accordance with ethical guidelines stated in Guide forthe use and care of laboratory animals, approved by the Committee on the Care and Use of Laboratory Animals of the Institute ofLaboratory Animal Resources Commission on Life Sciences, National Research Council, China (1985). DMEM/F12 serum-free medium,B27 additives, epidermal growth factor, basic fibmblast growth factor, and fetal bovine serum (volume fraction:0, 1) were purchased fromGibco Company, British; Interleukin lα, interleukin 11, and glial cell-derived neurotrophic factors were purchased from R&D Company,USA; In addition, leukaemia inhibitory factor (Perpotech, British), tyrosine hydroxylase(Santa Cruz, USA), nidogen antibody,microtubule-associated protein 2 antibody, and glial fibrillary acidic protein antibody(Chemicon, USA) were also used.METHODS: Six rats were randomly selected at each time point (on days 10,12,14,16, and 18 after gestation). After anesthesia, therats were sacrificed. Under the aseptic condition, fetal rat was harvested. Rat mesencephalic ventral brain tissue was isolated forculture of neural stem cells. Different gestational ages of rat brain-derived neural stem cells were separately cultured in theserum-free medium containing epidermal growth factors and basic fibroblast growth factors. After passage and amplification, theneural stem cells were induced to differentiate towards dopaminergic neurons in the medium containing interleukin lu, interleukin11, leukaemia inhibitory factors, glial cell-derived leukaemia inhibitory factors. On day 6 after induction and differentiation, thedopaminergic neurons were observed and identified by immunocytochemistry. After labeled by tyrosine hydroxylase, thedifferentiated dopaminergic neuron proportion was detected by a flow cytometer.MAIN OUTCOME MEASURES: The growth state of differentiated rat neural stem cells at different gestational ages and theimmunocytochemistry results. The tyrosine hydroxylase staining-positive neural stem cell proportion after induction anddifferentiation.RESULTS: Rat mesencephalic neural stem cell spheres on days 10,12, 14, 16, and 18 after gestation adhesively grew in thedifferentiation-inducing medium. The neural stem cells in the spheres gradually grew in radial tendency. On day 6 afterdifferentiation, most of the neural stem cells exhibited 1-2 long processes or several short processes. After nidogenimmunocytochemical staining, most of neural stem cells exhibited cytoplasm-positive. After culture for 6 days in the differentiationinducing medium, rat mesencephalic neural stem cells at gestational 10,12, 14, 16, and 18 days were detected by a flow cytometer.Results demonstrated that the proportion of tyrosine hydroxylase-positive cells was (10.3±2.5)%, (21.6±3.4)%, (16.7±2.8)%,(14.2±3.2)%, and (8.9±1.8)%, respectively. There was a significant difference in the proportion of tyrosine hydroxylase-positivecells among the cells at different gestational days (P < 0.05). Rat neural stem cells at gestational 12 days could be induced todifferentiate into dopaminergic neurons at the highest proportion.CONCLUSION: Mesencephalic neural stem cells of rats at different gestational days have different capabilities to differentiatetowards dopaminergic neurons. The proportion of dopaminergic neurons is the highest when mesencephalic neural stem cells ofrats at gestational 12 days.

BACKGROUND: Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of Parkinson's disease rats; however, the survival rate of transplanted cells has been low. Most cells die of apoptosis as a result of the formation of oxygen free radical and lipid peroxidation.OBJECTIVE: To observe resveratrol (Res) effects on survival of transplanted cells, transplanted efficacy and dopaminergic differentiation from neural stem cells in a rat model of Parkinson's disease.DESIGN, TIME AND SETTING: Randomized controlled animal experiments were performed at the Animal Experiment Center,Sun Yat-sen University from October 2007 to June 2008.MATERIALS: Thirty-two adult, healthy, male Sprague Dawley rats were equally and randomly assigned to model control, dopaminergic neuron, Res and combination groups. Four healthy Sprague Dawley rat embryos at gastational days 14-15 were selected and fetal rats were used for isolation and culture of neural stem cells. Res (Jingmal Biotech, Shenzhen, China) was used for this study.METHODS: Neural stem cells derived from the mesencephalon of embryonic rats were isolated and cultured in vitro, and passaged in serum-free culture medium containing epidermal growth factor and basic fibroblast growth factor, and then differentiated into dopaminergic neurons in differentia.ion medium. Parkinson's disease rat models were established by the injection of 6-hydroxydopamine in each group. Rats in the dopaminergic neuron group was injected with 3 pL cell suspension (1×10 cells/μL) containing dopaminergic neurons in the corpus striatum. Rats in the Res group received 3 μL of Ras (40 mg/L).Rats in the combination group were subjected to 3 μL of Res (40 mg/L) + 3 μL cell suspension (1×105 calls/μL) containing dopaminergic neurons. Rats in the model control group received 3 ×L of DMEM/F12 culture medium.MAIN OUTCOME MEASURES: The percentage of tyrosine hydroxylase-positive neurons in differentiated cells. The alteration of rotational asymmetry and the survival of tyrosine hydroxylase-positive neurons in graft areas of Parkinson's disease rats after transplantation.RESULTS: Flow cytometry demonstrated that survival rate of tyrosine hydroxytase-positive neurons was (17.8 ±4.2)% at 6 days following differentiation. Compared to the model control group, the rotational asymmetry was significantly improved at 10 days (P < 0.01), was significantly decreased at 20 days following transplantation in the combination group (P < 0.01). At 10-60 days following transplantation, the number of rotational asymmetry was significantly lower in the combination group than in the dopaminergic neuron group (P < 0.01). Tyrosine hydroxylase-positive neurons were not determined in the Res and model control groups. The number of tyrosine hydroxylase-positive neurons was significantly more in the combination group than in the dopaminergic neuron group (P < 0.01).CONCLUSION: Res can increase survival rate of transplanted cells in the corpus striatum, and improve rotational asymmetry in rat models of Parkinson's disease following transplantation of dopaminergic neurons differentiated from neural stem cells.Ke CL, Chen BL, Yu ZH, Huang ZS.Effects of resveratrol on the transplantation of neural stem call-derived dopaminergic neurons in a rat model of Parkinson's disease.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2009;13(27): 5281-5285.[http://www.crter.on http://en.zglckf.com]

Objective To investigate a safe and effective method of lengthening the short nose.Methods Thirty-nine cases of short nose were corrected by the bilateral septal spreader graft and columellar strut using autologous cartilage,combined with expanded polytetrafluoroethylene (e-PTFE)to reconstruct the supporting structures to improve the forward and upward strength of the nasal tip in order to increase the nasal length and to correct the over rotation of nasal tip.The shield and cap grafts were also used for the patients whose nasal tip were too low with vertical dome division technique.Results All the patients showed preserved nasal length after surgery with well-proportioned nasal features and the nasal lip angles were normal.There was no evidence of postoperative nasal shortening.No operative or postoperative complications were observed by follow-up for 6 months to 1 year,except for 2 cases with graft cartilage being apparent on nasal tip.All the patients were satisfied with the surgical results achieved.Conclusions It is necessary to provide powerful forward and upward strength to lengthen nose and correct the over rotation of nasal tip effectively and safely.The application of bilateral septal spreader graft and columellar strut using autologous cartilage and combined with e-PTFE represents a valuable tool for the short nose and proper cartilage grafts of nasal tip can improve the height of the nasal tip and further correct the over rotation of the nasal tip.

Objective Controversies remain as to the recovery time, recovery quality, and incidence of peri-recovery adverse reactions of the patient receiving general anesthesia with remifentanil and sufentanil.This study aimed to systematically assess the qual-ity of recovery from general anesthesia with remifentanil and sufentanil. Methods Randomized controlled trials ( RCTs) were re-trieved from The Cochrane Library, PubMed, MEDLINE, EMbase, Ovid, Springer, Web of Science, CNKI, CBM, VIP, and Wan-Fang Data.According to the modified Jadad quality scale, the literature retrieved was screened and subjected to quality evaluation and meta-analysis was performed on the included studies using the RevMan 5.1 software. Results Thirty-three RCTs involving 2175 pa-tients were finally included.The results of meta-analysis showed no significant differences between the remifentanil and sufentanil groups undergoing thyroid surgery either in the recovery time to spontaneous breathing ( T1) and eye-opening ( T2) and endortracheal extubation time ( T3 ) ( P >0.05 ) or in the incidence rate of postoperative nausea and vomiting (PONV) (P>0.05).T1, T2 and T3 were significantly shorter in the sufentanil than in the remifentanil group in other types of surgery (P<0.05).The patients with level-1 Ramsay score were remarkably more in the remifentanil than in the sufentanil group ( MD =13.67, 95% CI 2.67 -69.91 ) ( P <0.05), and the VAS scores were markedly higher in the former than in the latter group at 30 minutes (MD=3.37, 95% CI 3.28-3.46) and 1 hour after extubation (MD=2.53, 95% CI 2.43 -2.63) (P<0.05). Conclusion Compared with sufentanil, remifentanil provides a quicker recovery but a weaker analgesia effect and a higher rate of post-operative agitation.However, sufentanil produces a better pain relief and a higher quality of recovery after operation.

Aim To investigate the antagonistic effect of resveratrol on neuropathic pain and its underlying mechanism. Methods Neuropathic pain was induced by ligation of L5 spinal nerve (SNL) in rats. 90 male Sprague-Dawley rats, fit with intrathecal catheters were divided randomly into six groups ( n = 15 ): naive group; sham group; SNL group; high dosage of res-veratrol group (300μg);middle dosage of resveratrol group ( 30μg ) and low dosage of resveratrol group (3μg). The naive group did not make any process. In sham group, the L5 spinal nerve was only exposed without ligation. Other groups received SNL. Different dosages of resveratrol dissolved in 10μL 100% DMSO were administered by intrathecal injections once a day for 4 days, starting on day 4 after SNL. Paw withdraw-al latency (PWL) was measured on day 1,3,5,7,9, 11,14 days after surgery separately. On day 7 after be-havioral testing, the lumbar segments of the spinal cord were removed to measure the level of SIRT1 and acety-lated-p65(Ac-p65) for western blot. The activation of NF-κB was determined through calculating the percent-age of NF-κB-immunofluorescence positive staining cells in this study. Results Compared with sham groups,the SNL group showed an obvious decrease(P< 0. 05) of PWL and SIRT1 after surgery,whereas Ac-p65 and actived NF-κB significantly increased ( P <0. 05) in the spinal cord. Administration with high and middle dosages of resveratrol markedly attenuated(P <0. 05) SNL-induced thermal hyperalgesia and down-regulation of SIRT1 and blocked (P < 0. 05) the SNL-induced up-regulation of Ac-p65 and actived NF-κB in the spinal cord. Conclusion Intrathecal resveratrol can inhibit the development of neuropathic pain and suppress the activation of NF-κB signaling in SNL rats . The analgesic effect of resveratrol is implemented partly via increasing the level of SIRT1 and deacetylat-ing p65.

Objective To explore the effects of intrathecal injection of acetyltransferase p300 inhibitor Garcinolon on hyperalgesia in a rat model of L5 spinal nerve ligation and its underlying mechanism. Methods After lumber intrathecal catheters implanted,90 male Sprague-Dawley rats (40-50 d,weighing 180-220 g)were randomly divided into six groups (n=15 each):Naive group (group N),Sham operation group (group S),SNL group (group C)and three Garcinol treatment groups (group H:500μg/kg,group M:100μg/kg,group L:20μg/kg).Group N did not receive any operation in rats.In group S,the L5 spinal nerve was only exposed without ligation.Other four groups all received spinal nerve ligation (SNL).Group N,group S and group SNL were administrated intrathcelly with 100% DMSO (10 μl)within 3 to 6 days after SNL surgery.Other three groups were treated with Garcinol 500μg/kg (group H),100μg/kg (group M),20 μg/kg (group L)at the same points respectively.Behaviorally,thermal withdrawal latency (TWL) were measured at 1 day (T0 )before and on days 1(T1 ),3(T2 ),5(T3 ),7(T4 ),9(T5 ),11(T6 ),14(T7 )after surgery separately.On the 7th day after behavioral testing,the lumber segment of the spinal cord was removed to test the level of p300 and acetyl-p65 by western blot,while NF-κB was detected with immunofluorescence. Results Compared with group N,TWL was significantly shortened in group C,L,M and H,the levels of ace-tyl-p65 and p300 in group C,L,M and H were markedly increased,the expression of NF-κB in group C,L,M and H was markedly increased(P<0.05).Compared with group C,TWL was significantly prolonged in group M and group H,the levels of acetyl-p65 and p300 in group M and group H were dramatically decreased,the expression of NF-κB in group H was obviously decreased(P <0.05).Conclusion The acetyltransferase p300 inhibitor Garcinol imposes protective effect in SNL-induced neuropathic pain.Mechanisms are probably associ-ated with decreasing acetyl-p65 protein expression level in the NF-κB pathway.

Objective To evaluate the distribution and significance of IgG subclasses of anti-cyclic cirullinated peptide antibody (anti-CCP) in sera from patients with rheumatoid arthritis (RA).Methods A total of 83 patients with RA at the Department of Endocrinology of Taizhou Hospital , 51 disease controls and 50 healthy controls during the period from August 2012 to June 2013 were enrolled.The total serum IgG and IgG subclasses of anti-CCP antibodies were detected by antigen specific enzyme linked immune-sorbent assay( ELISA ).The prevalence and relative amount of IgG subclasses were calculated and compared.Statistical analysis was performed by χ2 test and Kruskal-Wallis H test.Results The positive rates of IgG subclasses of anti-CCP were anti-IgG 71.1%(59/83), anti-IgG1 78.3%(65/83), anti-IgG2 26.5%(22/83), anti-IgG3 60.2%(50/83), anti-IgG4 74.7%(62/83) respectively.The diagnostic value of anti-CCP-IgG1, anti-CCP-IgG3 and anti-CCP-IgG4 alone or combined (AUC =0.818-0.901),compared with anti-CCP-IgG(AUC=0.857), had no significant difference(Z=0.028-0.045,P>0.05).The DAS28 score of anti-CCP-IgG1(DAS28 =6.5), and anti-CCP-IgG4(DAS28 =6.5)positive in patients with RA were significantly higher than those in negative groups (DAS28=4.5,4.6)(U=396.0,427.5,P<0.01).The T28(T28=4.0,4.0)and SW28(SW28=4.0,4.0) results of CCP-IgG1and CCP-IgG4 positive in patients with RA were significantly higher than those in negative groups (T28=3.0,3.0,SW28 =3.0,3.0)(U=377.5,406.0,255.5,286.5,P<0.05).Conclusions The distribution of IgG subclasses of anti-CCP in sera from patients with RA was predominantly anti-CCP-IgG1, anti-CCP-IgG3 and anti-CCP-IgG4 associated with RA disease activity.However , whether joint detection of IgG subclasses can replace conventional anti -CCP is questionable.

Objective To observe the effects of nAChR antagonistα-conotoxin Eb1.6 on ther-mal pain threshold and spinal IL-1βexpression levels and astrocytes activation in rats using L5 spinal nerve transaction (SNT)model.Methods Fifty male Sprague-Dawley rats were randomly assigned into 5 groups with each group 10 rats:sham group,different doses of α-CTX Eb1.6 (0.1 5,1.5 and 1 5 nmol/kg)groups and the saline group after SNT.Saline solution or different doses of Eb1.6 were intraperitoneally injected seven days after the surgery when the model was stable and the treatment continued for seven days.Measured the TWLs of all groups of the rats 1,2,4,7,12 hours after the in-jection on 7 d and 13 d.The rats were sacrificed and L5 spinal cord tissues were collected immediately after the behavioral tests on 13 d.The expression of GFAP and IL-1βwere assessed by Western blot assay and enzyme linked immunosorbent assay (ELISA)separately.Results Groups E1,E2,E3 and C had shorter TWL before the injection on 7 d and 13 d than group N(P <0.05).The TWLs of the rats in groups E1,E2 and E3 of 1 h,2 h and 4 h after the injection on 7 d were significantly higher than that before the injection(P <0.05)with 2 h after the injection showed the most obvious change.The TWL of 1 h,2 h,4 h and 7 h after the injection of the rats in group E1,E2 and E3 and those of 12 h after the injection of the rats in group E2 and E3 on 13 d were significantly higher than that before the injection(P <0.05 )and also higher than TWL of the respective time points on 7 d(P < 0.05 ),also with 2 h after the injection showed the most obvious change.The TWLs of 2 h after the injection a-mong group E1,E2 and E3 showed significant differences both on 7 d and 13 d (P <0.05).Rats spi-nal IL-1βand GFAP expression levels of group E1,E2,E3 and C were significantly higher than those of group N(P <0.05).Rats spinal IL-1β and GFAP expression levels of groups E1,E2,E3 signifi-cantly decreased compared with group C(P <0.05).There were significant differences among the spi-nal IL-1βand GFAP expression levels of group E1,E2 and E3(P <0.05).Conclusion Eb1.6 dose-de-pendently reduced the thermal hyperalgesia induced by L5 spinal nerve transection.Repeated treat-ment of Eb1.6 could produce better analgesic effect,which might be partly attribute to the inhibition of spinal IL-βlevels and astrocytes activation.

Objective To evaluate the clinical effect of the facial soft tissue defect treated with platelet-rich plasma (PRP) mixed with purified fat.Methods 35 patients with facial soft-tissue defects were treated by PRP mixed with centrifuged fat tissue.Using Fuzzy judgment method the clinical effectiveness of therapeutic outcomes were evaluated on fullness,injection of frequency and satisfaction during 3 to 6 months of follow-up.Results The therapeutic outcomes showed that the reconstructed face became a relative symmetrical and natural appearance with not evidently fat resorption and stable after operation.Both patients and surgeons were satisfied with the results.The score of effectiveness was highest in Fuzzy judgment.Conclusions The efficacy of PRP is significant in enhancing fat graft,reducing fat necrosis and resorption.The fat tissue mixed with PRP is a reliable reconstruction option for the facial soft tissue defect.