The Role Of Vascular Endothelial Growth Factor On Spinal Motor Neuron And Its PI3/K Signaling Pathway

Part I The spinal motor neuron culture in vitroObjective To explore the method of spinal cord motor neurons cultured of Sprague-Dawley (SD) rats in vitro, which is for the further study of spinal cord motor neurons diseases.Methods Spinal cords were collected from neonatal SD rats and dissociated into cells in sterile conditions, purified by gradient centrifugation of Metrizamide.Motor neurons were cultured in Leibovitz's L-15 media, observed and identify by immunofluorescence.Results Spinal motor neurons cultured over 12 hours were adhesion, till 6 days, they turned to full size of cell body and clear synapse. More than 2 weeks after implantation, part of neuron died. Stained by SMI-32 to identify as spinal motor neurons.Conclusion We established the methods of spinal motor neuron culture in vitro of a higher purity. The procedure is relatively simple and in a high survival rate.Partâ…¡The role of VEGF in cultured motor neurons in vitroBackground Numerous studies on animal models and human showed the relationships between the low level of VEGF and motor neuron degeneration, not only in pathophysiology, but also in clinical manifestations. However, the clinical trial on exogenous VEGF as a treatment did not show a good outcome, even halting or delaying the course. In vivo studies, exogenous VEGF has a biphasic regulation of endogenous expression of the role of VEGFR.Objective To identify the role of exogenous VEGF on cultured motor neurons in vitro and to investigate relationship between the different doses and amounts. Design/Methods The cultured motor neurons of neonatal Sprague-Dawley rat in vitro, growed up to 6 days, were divided into control group and VEGF groups, which were treated with different doses of VEGF. All of them were observed by immunofluorescence and the viability of motor neuron by MTT.Using Ipwin used as an image analysis software for data collection and statistical analysis by SPSS 11.5.Results Compared with control group,there was a statistically difference in number and viability of VEGF (low/moderate) groups. VEGF helped to promote cell survival. However, VEGF(high dose) group showed no significant increase in both aspects.Conclusion The role of VEGF is to increase the rate and viability of cultured motor neurons with a low or moderate concentration, but has no effect with a high concentration.Partâ…¢The effects of VEGF on glutamate-induced impairment of motor neurons and its PI3/K expressionObjective To explore the moderate concentration of glutamate to induce impairment of motor neurons in vitro. And to study the effects of VEGF on glutamate-induced motor neurons impairment and its PI3/K expression.Method Spinal motor neurons were dissociated and cultured for 4d and then exposed to Glu (20mmol/L), Glu (5mmol/L) or Glu (0.5 mmol/L) for 48hrs, while the motor neurons in control group were exposed to only growth media throughout the experiment. To find out the appropriate glutamate concentration by MTT test.Spinal motor neurons were dissociated and cultured for 4d and then exposed to VEGF (1mmol/L), VEGF (0.1mmol/L) plus Glu (0.5 mmol/L) as Glu groups, plus to PI3/K inhibitor LY29004 as inhibitor group, exposed to only VEGF(1mmol/L) as VEGF group and exposed to only growth media as control for 48hrs. Viability of motor neurons was assessed after 48hrs.Motor neurons was assessed 48hrs after group by MTT, while PI3/K expression was observed by immunofluorescence and dectected by ELISA. All data was collected and statistical analyzed by SSPS 11.5.Result 1) Compared motor neurons viability of the control group with that of Glu groups, moderate and high concentrations of Glu groups could damage cell viability in a dose-dependent manner (P<0.05).2) It was found VEGF could inhibit glu-induced impairment of cell viability and also in a dose-dependent manner, to compared viability of motor neurons in each groups(P<0.05).However, PI3/K inhibitor LY294002 could reduce the effect of VEGF(P<0.05). Similiar findings was also discovered in comparison of PI3/K expression among different groups by immunofluorescence and ELISA detection.Conclusion 1) The cultured motor neurons in vitro with the increasing concentration of Glu reduced viability.2)Low or moderate concentration of VEGF promoted the cell viability of spinal motor neurons and also PI3/K expression. However it can not to glutamate-induced toxicity in viability, with reduction of PI3/K expression.lt was found VEGF could improve Glu-induced impairment of cultured motor neurons, stimulating the PI3/K expression and in a certain concentration range it was a dose-dependent effect. the LY294002, as a PI3/K inhibitor, decreased viability of the cultured motor neurons drastically, while it inhibits PI3/K expression. It counteracted the effect of VEGF in motor neurons.Conclusion1) Established the spinal motor neurons in vitro culture methods from neonatal SD rat with a high purity and in a relative simple way.2) The role of VEGF is to increase the rate and viability of cultured motor neurons with a low or moderate concentration, but no effect with a high concentration. In a word, the effects of exogenous VEGF on spinal motor neurons depends on concentration.3) Glutamate could decrease the survival rate of cultured motor neurons in vitro, which was negative correlated to the concentration. What is more, neither low nor moderate concentration of VEGF could block the damage.4) VEGF of low or moderate concentration could activates PI3/K expression in a dose-dependent manner.5) VEGF of low or moderate concentrations partially protect motor neuron from glutamate-induced excitatory amino acid toxicity, increasing the survival rate and PI3/K expression of motor neurons. 6) The PI3/K antagonist, LY294002 weakened the effect of VEGF on the viability and PI3/K expression, as is mentioned above.