| Apoptosis is an important event in the sub-acute stage of ischemic cerebro-vascular disease and it determines the prognosis in a certain extent. Compared with necrosis, apoptosis is a relatively progressive process. Thus, the way to protect the neurons from apoptosis is of great importance in the therapy of such diseases.Apoptosis is the result of a series of metabolic changes after ischemia. The recent studies attribute it to two distinct mechanisms: excito-toxic effect, which relies on the toxicity of excitatory amino acid (mainly glutamate), and non-excito-toxic effect, which is independent of that toxicity. These two mechanisms can alter the functions of mitochondria, activate the pro-apoptotic members of Bcl-2 family, and induce the proteolytic activation of the caspase family members via different routes. The destination of all the changes is neuronal apoptosis.Neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family. It is now convinced that NGF and brain derived neurotrophic factor (BDNF), two relatives in the same family, are up-regulated in hippocampus after transient forebrain ischemia and they can exert a protective effect on the neurons in penumbra via transcription dependent and transcription independent pathway. However, as to the effect of NT-3, contradictions arise. On one hand, the expression level of NT-3 is suppressed during ischemia; on the other hand, several experiments on the protective effect ofheterogeneous NT-3 have got different and in some cases entirely opposite results. Some experiments even show that NT-3 can do nothing but accentuate the injury ischmia imposes on neurons.To understand the real meaning of NT-3 expression on neuronal apoptosis after ischemia, we first observed the changes in NT-3 expression and neuronal apoptosis in hippocampus of rat during forebrain ischemia and reperfusion by in situ hybridization and TUNEL. We also analyzed the relationships between the two. These results show that in the areas of CA1, CA2, CA3 and dental gyrus, the expression level of NT-3 mRNA really decreases during transient forebrain ischemia and reperfusion. But in different regions, the decrease presents different character. The severest change both in duration and degree is found in CA1 while the modest one in dental gyrus and it is very interesting to realize that CA1 also show the greatest vulnerability to ischemic injury, while dental gyrus the modest. Statistic analysis shows that in each area, the level of NT-3 expression correlates well with the number of the apoptotic neurons. These suggest that the decrease in NT-3 expression may be responsible at least in part to neuronal apoptosis.In order to exclude the influences the complicated in vivo situation exerts on neuronal apoptosis, we made use of in vitro cultured hippocampal neurons to establish two types of injury model: oxygen deprivation and glutamate injury, to represent respectively the non-excito-toxic mechanism and excito-toxic mechanism. Adenovirus vectors were used to deliver the heterogeneous NT-3 cDNA to the cultured neurons, and the expression status of both heterogeneous and homogeneous ones during the process of injury was monitored by Western blotting. By means of immuno-histochemistry and TUNEL, the effects of NT-3 on Bcl-2 expression and neuronal apoptosis were detected. The results show that, in vitro cultured hippocampal neuronsexpress both NT-3 and Bcl-2 in a certain level while oxygen deprivation and glutamate toxicity suppress the expression procedure. Adenovirus vectors can successfully deliver the heterogeneous NT-3 cDNA into the hippocampal neurons in culture and support its correct translation and modification. The increase in NT-3 induces the recovery of Bcl-2 content and protect the neurons from apoptosis. The protective effect is accomplished partly by the upregulation of Bcl-2 expression.
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