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OBJECTIVE: Functional and neural tissue recovery has been reported in many animal studies conducted with stem cells. However, the combined effect of cytokines and stem cells has not yet been adequately researched. Here, we analyzed the additive effects of granulocyte colony-stimulating factor (GCSF) on adipose-derived stem cells (ADSCs) infusion in the treatment of acute spinal cord injury (SCI) in rats. METHODS: Four days after intrathecal infusion tubes implantation in Sprague-Dawley rats, SCI was induced with an infinite horizon impactor. In the Sham group (n=5), phosphate-buffered saline was injected 3, 7, and 14 days after SCI. GCSF, ADSCs, and ADSCs with GCSF were injected at the same time in the GCSF (n=8), ADSC (n=8), and ADSC+GCSF groups (n=7), respectively. RESULTS: The ADSC and ADSC+GCSF groups, but not the GCSF group, showed significantly higher Basso-Beattie-Bresnahan scores than the Sham group during 8 weeks (p<0.01), but no significant difference between the ADSC and ADSC+GCSF groups. In the ladder rung test, all four groups were significantly different from each other, with the ADSC+GCSF group showing the best improvement (p<0.01). On immunofluorescent staining (GAP43, MAP2), western blotting (GAP43), and reverse transcription polymerase chain reaction (GAP43, nerve growth factor), the ADSC and ADSC+GCSF groups showed higher levels than the Sham and GCSF groups. CONCLUSION: Our analyses suggest that the combination of GCSF and ADSCs infusions in acute SCI in the rat does not have a significant additive effect. Hence, when combination agents for SCI stem cell therapy are considered, molecules other than GCSF, or modifications to the methodology, should be investigated.
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Animais , Ratos , Western Blotting , Terapia Combinada , Citocinas , Proteína GAP-43 , Fator Estimulador de Colônias de Granulócitos , Células-Tronco Mesenquimais , Modelos Animais , Reação em Cadeia da Polimerase , Ratos Sprague-Dawley , Transcrição Reversa , Traumatismos da Medula Espinal , Medula Espinal , Células-TroncoRESUMO
Objective To evaluate the role of conventional protein kinase Cγ (cPKCγ)/growthassociated protein-43 (GAP-43) signaling pathway in ketamine-induced apoptosis in hippocampal neurons of developing rats in an in vitro experiment.Methods Primarily cultured hippocampal neurons were seeded in culture plates at a density of 1×10.6 cells/ml and divided into 2 groups (n=10 each) using a random number table:control group (C group) and ketamine group (K group).Group C received no treatment.Ketamine was added with the final concentration of 300 μmol/L in group K.At 12 h of culture or incubation,the apoptosis in hippocampal neurons was detected by flow cytometry.The apoptotic rate was calculated.The expression of cPKCγ,GAP-43 and phosphorylated GAP-43 in hippocampal neurons was measured by Western blot.Results Compared with group C,the apoptotic rates of hippocampal neurons were significantly increased,and the expression of cPKCγ,GAP-43 and phosphorylated GAP-43 was down-regulated in group K (P<0.01).Conclusion The mechanism by which ketamine induces apoptosis in hippocampal neurons of developing rats may be related to inhibition of cPKCγ/GAP-43 signaling pathway activation in an in vitro experiment.
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Objective To investigate the expressions of brain-derived nerve growth factor (BDNF) and growth-associated protein 43 (GAP-43) in the hypothalamus of rats inflicted with restraint stress and their relationship with behavioral changes.Methods Forty male SD rats were divided into control group,restraint stress 7-day group,restraint stress 14-day group,restraint stress 21-day group according to the random number table,with 10 rats per group.Behavior changes were observed by open-field test,serum levels of corticotrophin releasing hormone (CRH) by enzyme-linked immunosorbent assay,and expressions of BDNF and GAP-43 in the hypothalamus by western blotting.Results Restraint stress 7-day group exhibited increases in spanning lattice times (50.0 ± 7.0),standing times (11.4 ± 2.1)and modification times (11.2 ± 2.7) compared with all other groups (P ＜ 0.05).Restraint stress 14-day group and restraint stress 21-day group showed significant decreases in spanning lattice times (35.5 ±7.5,29.4 ± 6.8),standing times (7.8 ± 4.9,5.6 ± 3.9) and modification times (6.7 ± 2.9,4.4 ±2.6) compared with control group (42.6 ± 5.4,8.9 ± 4.3,and 7.9 ± 3.0) (P ＜ 0.05).Restraint 14-day and 21-day groups showed significant increases in serum CRH level [(750.73 ± 123.68) pg/ml and (793.06 ± 115.84)pg/ml] compared with that in restraint stress 7-day group [(500.48 ± 88.71)pg/ml,P ＜0.05],but all were lower than (336.72 ±45.34) pg/ml in control group (P ＜0.05).Levels of BDNF and GAP-43 in the hypothalamus were the lowest in control group (0.672 ± 0.185 and 0.694 ±0.253).However,restraint stress increased the expressions of BDNF and GAP-43 in the hypothalamus,with the highest level in restraint stress 21-day group (1.357 ± 0.524 and 1.486 ± 0.679) (P ＜ 0.05).Conclusion Restraim stress can up-regulate BDNF and GAP-43 proteins in the hypothalamus,and lead to plasticity changes that may relate to stress-related behavior.
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BACKGROUND:Reconstruction of damaged brain tissue through cel transplantation has become a new way to treat cerebral infarction. In recent years, bone marrow mesenchymal stem cels have become the new darling in cel transplantation therapy. OBJECTIVE:To investigate the effect of ginkgo-damole injection combined with bone marrow mesenchymal stem cel transplantation to improve the neurological function of acute cerebral infarction rats and its mechanism. METHODS:Animal models of middle cerebral artery occlusion were made in rats using suture method, and then 60 rat models were randomly divided into control group, cel transplantation group and combination group. The control group was given intravenous injection of PBSvia the tail vein; the cel transplantation group was given intravenous injection of bone marrow mesenchymal stem cel suspension (2.5×109/L) via the tail vein; the combination group was given intravenous injection of bone marrow mesenchymal stem cel suspension (2.5×109 /L) and ginkgo-damole injection (2 mL/kg, once a day, totaly 5 days)via the tail vein. Modified neurological severity scores were recorded at 1, 3 days and 1, 2 weeks after transplantation. At 2 weeks after transplantation, expressions of brain-derived neurotrophic factor and growth associated protein 43 in the brain were detected using RT-PCR; cel apoptosis detected using MTT assay; BrdU positive cels counted using immunohistochemistry method. RESULTS AND CONCLUSION:There were no differences in the modified neurologic severity scores among the three groups at 1, 3 days after transplantation (P > 0.05), but the modified neurological severity scores in the combination group were lower than those in the cel transplantation group and control group at 1, 2 weeks after transplantation (P < 0.05). The expressions of brain-derived neurotrophic factor and growth associated protein 43 in the brain were significantly higher in the combination group than the other two groups at 2 weeks after transplantation (P < 0.05); compared with the other two groups, the number of apoptotic cels was less but the number of BrdU positive cels was higher in the combination group (P < 0.05). These findings indicate that the combination of ginkgo-damole injection and bone marrow mesenchymal stem cel transplantation can increase the expressions of brain-derived neurotrophic factor and growth associated protein 43 in the brain, inhibit cel apoptosis and improve neurological function in rats with cerebral infarction.
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Objective To evaluate the influence of sevoflurane anesthesia on the expression of growth-associated protein 43 (GAP-43) and neural cell adhesion molecule (NCAM) in hippocampal neurons of neonatal rats.Methods Thirty-six pathogen-free Sprague-Dawley rats,aged 7 weeks,weighing 15-20 g,were randomly divided into 4 groups (n =9 each) using a random number table:control group (C group),1.5％ sevoflurane 6 h group (L group),3％ sevoflurane 2 h group (H1 group) and 3％ sevoflurane 6 h group (H2 group).Group L inhaled 1.5％ sevoflurane in oxygen for 6 h.H1 and H2 groups inhaled 3％ sevoflurane in oxygen for 2 and 6 h,respectively.Group C inhaled 30％ oxygcn only.When the neonatal rats were 14 days old,the rats underwent Morris water maze test for 7 consecutive days.Place navigation and spatial probe tests were carried out.After the end of Morris water maze test,the rats were sacrificed,and the hippocampus was obtained for determination of the expression of GAP-43 and NCAM in hippocampal neurons.Results Compared with group C,the escape latency was significantly prolonged,the time of staying at the original platform quadrant was shortened,and the expression of GAP-43 was down-regulated in L,H1 and H2 groups,and the frequency of crossing the original platform was decreased in L and H2 groups.There was no significant difference in NCAM expression among the four groups.Conclusion The mechanism by which sevoflurane anesthesia decreases the cognitive function may be related to down-regulated expression of GAP-43,but not related to NCAM expression in hippocampal neurons of neonatal rats.
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Objective To investigate the effect of lornoxicam on the.neurological behavior change and the expressions of growth associated protein-43 (GAP-43) and nerve growth factor (NGF) in dorsal root ganglion (DRG) on the peripheral nerve chronic constriction injury (CCI) model of rats.Methods Fifty Wistar rats were randomly divided into four groups:normal control group (n =5),CCI model group (CCI group),normal saline control group (NS group),and lomoxicam therapy group (L group) ; CCI,NS,and L groups were subdivided into 3 groups according to the different postoperative interval:3,7 and 14 days (n =5 each subgroup),respectively.The right sciatic nerve of rat was to be chronic constriction injure.Group L was given the rat 1.3 mg/kg of lomoxicam every 12 hours; then,the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of the rats of CCI,NS and L groups were measured at the different postoperative interval (3,7 and 14 days).After that,the DRG to the injured sciatic nerves were harvested.Immunohistochemistry was used to examine the expressions of GAP-43 and NGF.Results Comparing with the normal group,the latencies of MWT and TWL at CCI,NS and L groups were significantly declined at the third,seventh and fourteenth day after surgery (P ＜ 0.05),and the expressions of GAP-43 and NGF in DRG of CCI,NS and L groups were significantly increased after surgery (P ＜ 0.05).Comparing with the CCI group,the MWT and TWL of group L were significantly increased at the same time subgroup (P ＜ 0.05).The expressions of GAP-43 and NGF in DRG of group L were significantly declined at the same time subgroup (P ＜ 0.05).Conclusions Lornoxicam could relieve the symptoms of heat and mechanical hyperalgesia after sciatic nerve chronic constriction injury.It proved that lornoxicam was effective in the therapy of neuropathic pain.
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Objective To observe the protective effect on retinal ganglion cells (RGC) and the safety of intravitreal injected acteoside in rats.Methods A total of 50 male Sprague Dawley rats with the weight of 190-210 g were used in this study.Fifteen rats were used for safety experiment of intravitreal injection of acteoside.The rats were divided into group A,B,C,control group and blank group,three rats in each group.The rats in group A,B and C were received intravitreal injection of 5 μl acteoside at the concentration of 1,2,and 5 mg/ml,respectively.Phosphate buffer solution (PBS) was injected in rats of control group.No treatment was performed for blank group.The retinal structure was examined by hematoxylin-eosin (HE) staining of retinal frozen sections at one,two and three weeks after injection.The retinal ultrastructure was examined by ultrathin section under transmission electron microscope at one and three weeks after injection.Others 35 rats were used for experiment of protective effect of acteoside on RGC.The rats were divided into operation group A and B (n=8),sham operation group C and D (n=8),and blank group (n=3).The optic nerve of rats in operation group was clamped for 10 seconds after optic nerve exposure,while the optic nerve of rats in sham operation group was exposed only.The rats in operation group A and B were received intravitreal injection with 5 μl acteoside (1 mg/ml) and 5 μl PBS respectively.The rats in sham operation group C and D were received intravitreal injection with 1 μl acteoside (1 mg/ml) and 1 μl PBS respectively.No treatment was performed for blank group.The retinal structure was examined by HE staining of retinal frozen sections at one,two and four weeks after injection.Immunohistochemistry was used to measure the expression of growth associated protein 43 (GAP-43).RGC apoptosis was assessed by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labelling (TUNEL) method.Software of SPSS 13.0 was used for the data statistical analysis in this study.Results In the safety experiment of intravitreal injected acteoside,there was no abnormity of cornea,anterior chamber,lens,vitreous cavity and retina after injection.At one,two and three weeks after injection,the retina structure was normal without significant apoptosis,necrosis and inflammatory cell infiltration.The ganglion cell layer showed slightly edema; there was no obvious change of retinal ultrastructure after injection of acteoside with 5 mg/ml and 2 mg/ml,but slight change with the format of 1 mg/ml.Transmission electron microscopy showed that intravitreal injection of 5μl acteoside at the concentration of 2 or 5 mg/ml can induce significant changes of micro-structures of retina,while injections at 1mg/ml can only induce minor changes.In the experiment of protective effect of acteoside on RGC,light microscope revealed that the cell showed typical changes of apoptosis in operation group,but not in sham operation group and blank group.At the first and second week after injection,compared with the sham operation group and blank group,the RGC number was decreased in operation group.The difference of RGC numbers between operation group A and B was statistically different (F=26.206,P＜0.05).The RGC numbers in operation group continues to decrease at the fourth week after injection,there was obvious difference compared with the first and second week after injection (F=17.364,P＜0.05),but there was no difference of RGC numbers among sham operation intra-group and between sham operation group and blank group at all the time points.Immunohistochemistry showed that at the first week after injection,the integrated absorbance (IA) value in operation group was higher than that in other groups (F=33.466,P＜0.05) ; there was no difference of IA value between operation group A and B.At the second week after injection,IA value in operation group A had slightly declined,but higher than that in operation group B (F=14.391,P＜0.05).At the fourth week after injection,IA value in operation group A declined further,but also higher than that in other groups (F=4.178,P＜0.05).TUNEL showed that on the first week after injection,RGC apoptosis rate in operation group was increased than that in other groups (F=15.365,P＜0.05).At the second week after injection,RGC apoptosis rate in operation group was decreased,and it in operation group A was lower than that in operation group B (F=15.365,P＜0.05).At the fourth week after injection,RGC apoptosis rate in operation group was decreased obviously,there was no difference compared with other groups (F =2.057,P ＞ 0.05).There was no difference of RGC apoptosis rate between sham operation group and blank group at all the time points.Conclusion Intravitreal injection of 5 μl acteoside (1mg/ml) is safe for rat retina,and can up-regulate GAP-43 expression and inhibit RGC apoptosis in optic nerve crush rats.
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Objective To observe the effects of voluntary exercise on the learning ability, memory and hippocampus growth-associated protein 43 (GAP43) expression in senescence-accelerated prone mouse (SAMP8), so as to explore the possible mechanism of exercises on improving the cognitive ability and delaying aging. Methods A total of 60 three-month old female SAMP8 mice were evenly assigned to running cage environment (RCE) group and standard environment (SE) group at random. After three months, Morris water maze test was used to test the platform-seeking latency and search strategy. Then 10 mice were sacrificed in each group for RT-PCR analysis of hippocampus GAP43 mRNA expression, 10 for Western blotting analysis of hippocampus GAP43 protein expression, and 10 for immunohistochemistry staining of hippocampus GAP43 expression. Results Morris water maze test showed that RCE mice had a significant shorter platform-seeking latency than SE mice(P<0. 01, P<0. 05), and RCE mice had a significant longer time in the first quadrant (P<0. 01) and a shorter time in the fourth quadrant (P<0. 05) compared with SE mice. RCE mice had a significantly higher GAP43 expression in the hippocampus compared with SE mice (P<0. 01). Conclusion Voluntary exercise can improve the learning ability and memory of SAMPS, which might be associated with the increase of GAP43 in the hippocampus.
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Introducción: cerca del 5% de los pacientes con dengue hemorrágico pueden presentar manifestaciones neurológicas; sin embargo, existe poca información sobre la infección directa por el virus dengue (DENV) en neuronas. Objetivo: determinar el papel del fenotipo neuronal en la infección por DENV en células de neuroblastoma SH-SY5Y inducidas o no a la diferenciación con ácido retinoico (AR). Materiales y métodos: células SH-SY5Y fueron inducidas con AR a diferenciarse e infectadas con DENV. Posteriormente se cuantificó la expresión de antígeno viral y de dos marcadores de diferenciación (GAP43 y sinaptofisina). También se evaluó la viabilidad postinfección por la técnica de MTT. Resultados: se encontró que las células diferenciadas son más susceptibles a la infección por DENV, pues se detectó en ellas mayor cantidad de antígeno viral que en las indiferenciadas. A pesar de que el virus indujo muerte celular en ambos tipos de células, la proporción fue mayor en las indiferenciadas (40,3% frente a 21,5%). La infección por DENV en células SH-SY5Y diferenciadas indujo una disminución significativa en la expresión de GAP-43 y sinaptofisina. Conclusiones: los resultados que se presentan permiten sugerir una relación entre la infección viral y la función neuronal, que podría ser importante para esclarecer la patogénesis de las manifestaciones neurológicas durante las formas graves de dengue.
Introduction: Approximately 5% of patients suffering from dengue hemorrhagic fever may have neurological manifestations. However, little information is available about direct infection of neurones by dengue virus. Objective: To determine the role of neuronal phenotype during DENV infection in human neuroblastoma cell line SH-SY5Y, either induced or not to differentiate by treatment with retinoic acid (RA). Materials and methods: Neuroblastoma cell line SH-SY5Y was induced to differentiate with RA and infected with DENV. The expression of viral antigen and of two differentiation markers of neurones, GAP-43 and synaptophysin, was evaluated quantitatively. Postinfection viability was also evaluated by the MTT technique. Results: It was found that differentiated cells are more susceptible to infection by dengue virus since more viral antigen was found in them than in the undifferentiated ones. DENV infection caused death in both cell types, but the rate was higher in the undifferentiated ones (40.3% vs 21.5%). In addition, DENV infection in differentiated SH-SY5Y cells induced a significant decrease in GAP-43 and synaptophysin expression. Conclusions: These results allow us to suggest a relationship between DENV infection and neuronal function, which could be important to elucidate the pathogenesis of neurological manifestations occurring in severe dengue disease.
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Humanos , Neuroblastoma , Sinaptofisina , Tretinoína , Vírus da Dengue , Infecções , Neurônios/virologiaRESUMO
Objective To evaluate the effect of intraperitoneal (IP) clonidine on the expression of GAP-43 mRNA in the spinal cord in a rat model of chronic neuropathic pain. Methods Thirty-six male SD rats weighing 180-220 g were randomly assigned to one of 3 groups (n=12 each) : group Ⅰsham operation (S); group Ⅱ chronic constriction injury (CCI) and group Ⅲ tP clonidine + CCI (CL). The animals were anesthetized with IP 10% chloral hydrate 300 mg/kg. The right sciatic nerve was exposed and 4 ligatures were placed in group CCI and CL. Clonidine 1 mg/kg was given IP immediately after surgery in group CL. Paw-withdrawal threshold (PWT) to thermal and von Frey filament stimulation was measured before (T_0, baseline) and at 3, 7 and 14 days after surgery (T_(1-3)). The animals were then killed. The lumbar segment of the spinal cord was removed for determination of the expression of GAP-43 mRNA. Results The PWT to thermal and mechanical stimulation was significantly reduced at 3 days after surgery (T_1) in group CCI and CL as compared with group S, and was significantly higher at T_2 and T_3 in group CL than in group CCI. The GAP-43 mRNA expression in the spinal cord was significantly increased in group CCI and CL as compared with group S and significantly lower in group CL than in group CCI. Conclusion lntraperitoneal clonidine can inhibit hyperalgesia by reducing the expression of GAP-43 mRNA in the spinal cord in a rat model of chronic neuropathic pain.
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0.05).There was significant difference in the optical density of the 43kDa protein between the patients with OAB and controls(P
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Objective To examine and evaluate the ability of spontaneous regeneration following various extent of incomplete injury in adult rats. Methods The quantities and reduplicated various extent incomplete injured model of adult rats optic nerve was established using different wounding time with constant wounding force produced by across action forceps at 2mm behind eyeball. GAP-43 (growth associated protein-43) and its mRNA expressions were detected with immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). All data were analyzed by ANOVA. Results GAP-43 and its mRNA expression levels revealed that there was negative response at the distal area contrast to the strong positive response at the proximal in earlier period after injury until half mouth. One mouth later, the increased GAP-43 and its mRNA expression levels became more and more high and reached the climax at second month post injury. Then it decreased gradually. The result of RT-PCR showed there are significant difference among the various extent incomplete injured models and different time after injury. Conclusion Spontaneous regeneration of adult rat’s optic nerve can be detected and identified following incomplete injury, and the extent of regenerating ability is correlated with the extent of injury.
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Objective To discuss the effects of utero placental ischemia on the body and nervous system development in fetal rats Methods By clamping the unilateral uterine artery of the rat, we produced a utero placental ischemia model The opposite uterus of the rat with normal uterine artery supply served as control We compared the body weight, weight of brain, and the expression of growth associated protein 43(GAP 43) mRNA in cerebral tissue by RT PCR in the 13 day (group 1) and 17 day(group 2) old fetal rats respectively Results The body weight and weight of brain in group 1 were 3 2 g and 0 16 g respectively, significantly lower than those of control 1 of 3 6 g and 0 18 g respectively ( P 0 05) However, GAP 43 mRNA in cerebral tissue of the group 2 (1 06) was significantly decreased compared with that of its control(1 21 )( P
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Objective To compare activated and normal Schwann cells in GAP43 gene expression. Methods 10 male SD rats, weighed from 100 g to 120 g. The right median nerve of SD rats was transected at the axillary level and was buried in muscle for predegeneration. 1 week later, the distal segment of the transected right median nerve with 1 cm long was harvested. The untreated left median nerve was harvested as control with same length. The epineurium of nerve was stripped, then the nerve tract was cut to small pieces. Schwann cells were obtained by way of double kinases digestion with 0.25% trypsin and 0.03% collegenase. The right median nerve was activated with additional liquid during digestion so as to obtain the activated Schwann cells. The normal Schwann cells were harvested from left median nerve. rt-PCR was used for GAP43 gene enlargement. mRNA was distilled from activated Schwann cells and untreated Schwann cells respectively. Then the mRNA was reversely transcripted to cDNA with SuperScriptTM, and cDNA worked as template for PCR enlargement. The product of PCR was separated with 1% agarose gel electrophoresis for 40 -50 min and stained with SYBR Green Ⅰnucleic acid gel. Fluorescence intensity of GAP43 PCR products was measured and then compared between the experiment group and control group. Results The Fluorescence intensity of GAP43 PCR product of activated Schwann cells was higher than that of normal Schwann cell. There was significant difference (P=0.003, Paired t test). It indicated that GAP43 mRNA of activated Schwann cells was much more than that of the normal Schwann cells. Conclusion GAP43 gene expression is up regulated in activated Schwann cells in contrast to normal Schwann cells. Activated Schwann cells secreting more GAP43, which may be one of the important mechanisms in promoting nerve regeneration.
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AIM:To investigate the expression of GAP-43 mRNA and protein of the motor neurons in spinal cord following the brachial plexus avulsion injury.METHODS: In the present study,three kinds of models of brachial plexus avulsion injury were made: right C7 anterior root avulsion(group A),C7 anterior root avulsion with right C5-T1 posterior roots breaking(group B),right C7 anterior root avulsion with hemi-transect between C5 and C6 segment of spinal cord(group C).The expression of GAP-43 mRNA in anterior horn of spinal cord was detected at 14 days after operation by SYBR green quantification RT-PCR technique.The amounts of GAP-43 positive neurons in spinal cord were detected at 1,3,7 and 14 days after operation by immunohistochemistry technique.RESULTS: In control group,the expression of GAP-43 mRNA was very low in anterior horn.By 14 days after operation,the expression of GAP-43 mRNA was evidently up-regulated compared with control group.GAP-43 positive neuron was observed in control group at 1st day and 3rd day after operation.GAP-43 positive neurons appeared at 7th day and peaked at 14th day after operation.The expression of GAP-43 mRNA and protein were maximum in group C,group B was the lowest.CONCLUSION: The expression of GAP-43 mRNA and GAP-43 protein were up-regulated following brachial plexus injury.The expression of GAP-43 protein results from the recombination of proteins.GAP-43 is closely related to the axon regeneration and functional reconstruction.