| Objective: To establish hemitransection spinal cord injury (htSCI) model of Macaque and observe the changes in expression of epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) in ventral horn and right cortex motor area of quadrumana following htSCI. Furthermore, to explore the molecular mechanism for the expression of EGF and GDNF following htSCI, and to explore the nerve repair function acted by EGF and GDNF. Besides, to offer the datas for applying EGF and GDNF to accelerate the recovery after spinal cord injury.Method: 18 adult healthy macaques (provided by experimental animal faculty, Kunming Medical College), with body weight ranging from 5.5Kg to 6.0 Kg, were used. All of them were divided at random into six groups: sham-operational group (3, in which the dura matter of spinal cord was cut but the spinal cord remained intact), day 7, day 14, month 1, month 2 and month 3 spinal cord injury groups (3 each, in which the spinal cord was hemitransected in the left at T11), animals survived for 7 days, 14 days, 1 month, 2 months and 3 months after operation respectively). 3 monkeys were used in every group. The normative nursing project was employed to monkeys so as to preclude thecomplication and reduce the death rate following htSCI. The animals were put to death at the corresponding time after operation and 3 monkeys were used in each point. The spinal cord of each monkey was taken out between 5mm up to and 5mm down to the lesioned point. And right cortex motor area was taken out. The tissue was put into 4% paraformaldehyde for 12h, and then into 30% sucrose PBS over night. Then the tissue blocks were made into 25 P m frozen sections. Under the same condition using specific EGF and GDNF antibody, the immunohistochemistry SP method was performed on those sections. Datas for the number of positive neurons both EGF and GDNF in the ventral horn of spinal cord and the right cortex motor area were analyzed by one-way ANOVA and paired samples t test.Results: We analyzed the number of EGF and GDNF immunoreacivity positive neurons located in ventral horn of spinal cord and the right cortex motor area for sham-operational group, day 7, day 14, monthl, month 2 and month 3 groups.1. EGF immunoreactivity positive neurons:(1) ventral horn of spinal cord: EGF immunoreactivity can be observed in motoneurons of the ventral horn in the sham-operational group, and the labeling was mainly localized in cytoplasm. As to the SCI groups, the EGF positive neurons at lesioned side and intact side of rostral or caudal part of spinal cord for all the spinal cord injury groups were all decreased in number. And the differences compared with sham-operational group were all significant (P<0.05). In the intact side of the rostral part, the number of positive neurons localized in ventral horn decreased sharply on 7 day after htSCI. Until 14 day, the number of positive neurons a kept at a low level. One month later after htSCI, the number increased obviously. One more month later, the number of positive neurons decreased again, and it maintained at that level till 3 months after lesioning. In the lesioned side of the rostral part, the number of positive neurons decreased sharply on 7 day after htSCI, but it increased sharply on 14 day, and it kept atthis level till 1 month. On 2 month the number decreased obviously, and there was a trend of decrease on 3 month after htSCI. In the intact side of the caudal part, the number of positive neurons decrease sharply, it increased again till 14 day and reached the peak at the time after htSCI. It continuously maintained at this high level till 1 momth. But it decreased obviously till 2 month, and kept this trend till 3 month after htSCI. In the lesioned side of the caudal part, the positive neurons decreased obviously following htSCI, whereas it increased sharply till 14 day and reach to the peak. After this it maintained at the level till 1 month. On 2 month it decreased sharply, and kept the decline trend till 3 month.(2) right cortex motor area: EGF positive neurons can be observed in the right cortex motor area in the sham-operational group, and the immunoreactivity was mainly localized in the cytoplasm. As to the htSCI groups in number, the EGF positive neurons in right cortex motor area were all decreased. And the differences compared with sham-operational group were all significant (PO.05). The positive neurons decreased obviously on 7 day after htSCI. It increased apparently and reached the peak on 14 day. Thereafter the number of positive neurons was stable till 1 month, and it decreased sharply again on 2 month. It changed little till 3 month.2. GDNF immunoreacivity positive neurons:(1) ventral horn of spinal cord: GDNF immunoreactivity could be observed in motoneurons of the ventral horn in the sham-operational group, and was mainly localized in the cytoplasm. As to the htSCI groups, the GDNF positive neurons at lesioned side and intact side of upside or subside of spinal cord were all decreased. And the differences compared with sham-operational group were all significant (PO.05). In the intact side of the upside to the lesion, the number of positive neurons localized in ventral horn decreased sharply on 7 day after htSCI. And it kept decreasing till 14 day. One month after htSCI, the number increased obviously, and reached the peak. After this, the number of positive neurons kept decreasing till 3 month after lesioning. As to the lesioned side ofthe rostral part, the number of positive neurons decreased sharply on 7 day, and it remained stable till 14 day. It increased sharply until 1 month, thereafter it maintained at this level till 2 month. On 3 month the number decreased obviously. In the intact side of the caudal part to the lesion point, the number of positive neurons kept decreasing till 14 day, and reached the lowest point. It increased sharply and reached the peak till 1 month. Thereafter it decreased continually till 3 month. In the lesioned side of the caudal part, the positive neurons kept decreasing trend on 14d following htSCI, and it reached to the lowest at the time. Till 1 month, it brought up again. Henceforth, it kept the decline trend till 3 month.(2) right cortex motor area: GDNF positive neurons can be observed in the right cortex motor area in the sham-operational group, and the immunoreactivity was mainly localized in the cytoplasm. The positive neurons decreased sharply and reach the lowest point on 7 day after htSCI. Thereafter it increased slowly till 1 month, and rose obviously till 2 month. On 3 month it reached to the normal level on the whole.Conclusion: 1. It was not as that to the rat, for mokeys, the EGF and GDNF positive neurons decreased apparently in ventral horn of spinal cord near the lesion point during 3 months after htSCI, though sometimes the number of positive neurons was upregulated, it was less than the sham-operational group. 2. It affected the expression of EGF and GDNF for motor neurons in ventral horn in lesioned side as well as in intact side following htSCI. The results suggest that transecting one side of spinal cord to observe the regeneration as contral of the other side is not exactitude at certain instance in the study of repair for SCI. 3. We discovered that the number of EGF immunoreactivity-like neurons as well as the number of GDNF immunoreactivity-like neurons were both decreased sharply and then increased spontaneously early after spinal cord injury. It suggest that the best opportunity to therapy SCI is in the forepart following SCI. 4. Within 3 month following htSCI in the left, the number of GDNF or of EGFimmunoreactivity-like neurons changed inconsistent in right cortex motor area. It indicate that this two neurotrophic factors response differently in cortispinal motor neurons following SCI. And it also clue on that intricate remolding had happened in cortex motor area after SCI, of course, the change of neurotrophic factors is a part of the remold. The difference for the change of the two neurotrophic factors after SCI suggest that they perform their function differently in the central nervous system.
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