The Effect Of BDNF On Survival,Regeneration And Plasticity Of The Spinal Cord Anterior Horn Neurons

Purpose:this study discusses possible effect of endogenous BDNF on ventricornu neuron protection by structuring animal model of sciatic nerve injury of rat, giving BDNF antibody and endogenous BDNF and observing influences of endogenous BDNF on spinal cord anterior horn neurons. Method:Establish sham operation group, control group and experimental group by constructing animal model of sciatic nerve injury of rat. Experimental group enterocoelia is injected with BDNF antibody; control group enterocoelia, injected with equal amount of physiological saline; both two times per week. Select6rats for each group and execute them on the7th day and14th day respectively; adopt Nissl body dyeing and TUNEL dyeing; observe cell function of ventricornu neuron of executed rats and its apoptosis conditions; at the same time, adopt Western blot to check changes of Bax、Bc1-2、Caspase-3protein level in ventricornu neuron of rats. Results:7days after operation, Nissl body dyeing shows that neurons in sham operation group presents normal neurons while neurons in control group presents rounded swelling and Nissl body reduces; a large number of Nissl bodies in experimental group dissolves and cell nucleus displacement can be seen. The number of TUNEL dyeing positive neurons, Bax. and Caspase-3protein level in experimental group is strikingly higher than that in control group (NS) and sham operation group (P<0.05) while Bcl-2protein level is remarkably lower than control group (NS) and sham operation group. The difference enjoys statistical meaning (P<0.05).14days after operation, sham operation group has no obvious change while vacuolar degeneration can be seen in cytoplasm in control group, but a small amount of normal neurons can also be seen. Neuron Nissl bodies in experimental group are disintegrated and even disappeared; neuron structure is obscure and has little of survival neurons. The number of TUNEL dyeing positive neurons in experimental group increases by35%, which is remarkably higher than that in control group and sham operation group (P＜0.05). Compared with that of7days, Bax and Caspase-3protein level in both control group and experimental group increases at the same time (P<0.05) and Bcl-2protein level in experimental group is strikingly lower than that in control group, Bax and Caspase-3protein level is obviously higher than that in control group. The difference enjoys statistical meaning. Conclusion:BDNF may be participated in anti-apoptotic reaction process of neurons after sciatic nerve injury, which has protective effects on injured neurons. Purpose:this study analyzes whether BDNF can influence and adjust expression of ventricornu neuron GAP-43synapsin I and SYN so as to initially explore possible mechanism of endogenous BDNF stimulating regeneration and plasticity of nerve injury by constructing animal model of sciatic nerve injury of rat. Method:Establish sham operation group, control group and experimental group by constructing animal model of sciatic nerve injury of rat. Experimental group enterocoelia injects BDNF antibody; control group enterocoelia injects equal amount of physiological saline; both two times per week. Select6rats for each group and execute them by7days and14days respectively; adopt RT-PCR and Western blot to observe GAP-43, synapsin I and SYN RNA as well as protein changes within spinal cord anterior horn neurons. Results:a small amount of GAP-43RNA expressions in ventricornu neuron can be seen in sham operation group but no protein expression. A large amount of GAP-43, synapsin I, SYN RNA and protein expression can be seen in control group; GAP-43, synapsin I, SYN RNA and protein expression in ventricornu neuron in experimental group are obviously lower than that in control group (P<0.05). GAP-43, synapsin I, SYN RNA and protein expression in experimental group is much higher than that in sham operation group. The difference has statistical meaning (P＜0.05). The results from7days and14days are the same. After14days, GAP-43protein in control group is remarkably higher than that in7-day groups (P＜0.05). synapsin Ⅰ, SYN RNA and protein expressions in ventricornu neuron in various groups after7days and14days have no obvious difference (P>0.05). Conclusion:BDNF is helpful to the recovery of injured nerve functions and promotion of regeneration and plasticity of neurons by influencing synthesis and phosphorylation of GAP-43, synapsin Ⅰ and SYN within spinal cord anterior horn neurons. Purpose:This part studies influence of BDNF on spinal cord anterior horn neurons cultured in vitro through in vitro serum-free culture of spinal cord anterior horn neurons of rat. Method:select a Wistar rat which has already been pregnant for15days, take out embryo in its uterus, separate its spinal cord ventral, make it into cell suspension and culture it serum-free. If the cells are cultured outside cell body for seven days, divide them randomly into experimental group, BDNF group, BDNF anti-body group and control group. Add BDNF (its final concentration is40ng/ml), BDNF anti-body (its final concentration is30μg/ml) and equal amount of PBS liquid in culture solution. The cells are continued culturing for another five days. Use Immunohistochemistry to test identification of neuron-specific markers like NF-200, MAP-2and NSE on nerve cells, calculate quantity of nerve cells under microscope and observe neuron form (cell counting is30visual field under microscope, take the average value). Adopt RT-PCR to check synapsin Ⅰ of nerve cells in vitro and SYN RNA expression level; Adopt Western blot to check synapsin Ⅰ, SYN, GAP-43, Akt, phosphorylated Akt, bcl-2, caspase-3and bax protein expression level.Result: Immunohistochemistry results show that for above90%of cultured cells, the immunoreactions of NF-200, MAP-2and NSE are presented as positive. Counted under microscope, survived cells in control group, BDNF anti-body group and BDNF group are42.23±3.26,32.32±2.35and49.23±3.12respectively. Survived cells in BDNF group are remarkably higher than those in both BDNF anti-body group and control group (P<0.05) while Survived cells in control group are obviously higher than those in BDNF anti-body group. The difference enjoys statistic meaning (P＜0.05). Through morphologic observation, it is discovered that, compared with BDNF anti-body, nerve cell bodies in control group and BDNF group are larger and richer and cells are strikingly becoming more and more. It is discovered from results of RT-PCR and Western blot, synapsin Ⅰ, SYN mRNA and protein level in BDNF group are remarkably higher than those in control group and BDNF anti-body group (P<0.05) while synapsin I and SYN in control group are obviously higher than those in BDNF anti-body group. The enjoys difference statistic meaning (P<0,05). In these three groups, Akt protein expression has no significant variation; phosphorylated Akt and bcl-2protein level in BDNF group are significantly higher than those in control group and BDNF anti-body group (P＜0.05) while bax and caspase-3protein level in BDNF group are obviously lower than those in control group and BDNF anti-body group (P＜0.05). Phosphorylated Akt and bcl-2protein level in control group are significantly higher than those in BDNF anti-body group while bax and caspase-3protein level are obviously lower than those in BDNF anti-body group. The difference enjoys statistic meaning (P<0.05).Conclusion:on one hand, BDNF can regulate synapsin I and SYN expression within spinal cord anterior horn neurons of rat cultured in vitro, stimulate synapse development and maturity and participate in synapse reorganization, which is conducive to recovery of injured neuron function. On the other hand, BDNF can effectively prevent nerve cells from dying and can foster survival of spinal cord anterior horn neurons cultured in vitro.