| Objective: To observe the spatio-temporal changes of the expression of brain derived neurotrophic factor (BDNF) and its receptor (TrkB) in the ventral horn of the spinal cord after hemitransection injury of the spinal cord (hSCI) on adult rhesus monkeys; to explore the potential role and possible molecular mechanism of BDNF and TrkB after hSCI; to explore the nerve repair function acted by BDNF and TrkB; and to provide the anatomic data for applying BDNF and TrkB to accelerate the recovery after spinal cord injury.Method: Twenty-one healthy, adult rhesus monkeys of Yunnan, China, provided by Experimental Animal Center, Kunming Medical College, with body weight ranging from 5.5 to 6.0 kg, were used in the experiment. All of them were divided at random into 2 groups: spinal cord injury(SCI) group (n=18, in which the left half of the spinal cord was hemitransected at T8) and sham-operational control group (n=3, in which the dura matter of the spinal cord was cut but the spinal cord remained intact and the animals were sacrificed after 24h). In the SCI group, the animals were divided further into 6 subgroups: 2d, 7d, 14d, 30d, 60d and 90d groups according to their survival time after operation respectively. There were 3 monkeys in each subgroup. In terms of a modified Tarlov's method of evaluation, the hind limb motor function of the 90d group animals was checked and evaluated consecutively on different time points. The normative nursing project was employed to monkeys so as to preclude the complication and reduce the death rate following hemitransection. The animals were put to death at the corresponding time after operation. The animal was perfused with 4% paraformaldehyd solution through aorta. The T8 spinal cord was taken out and fixed further with the same fixative, and then was kept in 20% sucrose solution over night. The thoracic cord tissue blocks were cut into frozen sections with a cryostat. Under the same condition, the immunohistochemistry SP method was performed on two sets of those sections by using specific BDNF and TrkB antibody respectively. The expression changes of positive BDNF and TrkB immunoreactivities in the ventral horn of the thoracic cord below the operative incision were observed. Data of them were analyzed by one-way ANOVA.Results: 1. The motor function of the left hind limb was evaluated as 0 level 2 days after operation. The muscular tension was decreased and a soft paralysis was found. The right hind limb was 2-3 levels. Seven days after operation, the left hind limb was 0-1 levels, and the right hind limb gradually recovered to 4 levels and kept till the 90th day we checked. Fourteen to thirty days after operation, the left hind limb recovered to 1-2 levels. Sixty days after operation, the left hind limb was 2 levels and a light grasping action of the toes was found. Ninety days after operation, the left hind limb recovered to 2-3 levels and the small joint motion was more nimble.2. In the ventral horn neurons of the thoracic cord gray matter in each animal group, the positive BDNF and TrkB immunoreactive products showing buffy granules were found. Positive BDNF immunoreactive products were mainly in the cytoplasm. The cytoplasm was stained deeply and had a demarcation with its nucleus, which was not stained. A strong positive TrkB immunoreactivity was also seen in the cytoplasm, but a diverse staining in its nucleus was found in the different positive TrkB immunoreactive neurons.3. After hSCI, (1) the number of the positive BDNF and TrkB immunoreactive neurons in the ventral horn of the thoracic cord gray matter in each animal group was decreased significantly comparing with the sham-operational control group (p<0.05). Moreover, from 7d to 90d group, there were an obviously similar tendency in expression changes of BDNF and TrkB. (2) The number of the positive BDNF neurons was decreased definitely in 2d group, and reached the lowest level in 7d group. In 14d group the number began to increase, and kept increasing to a peak level in 30d group. At this time point, the number of the positive BDNF neurons did not recover to the normal level and was still less than the one in sham-operational control group. From 60d to 90d group, the number showed a continually decreasing trend. (3) The number of the positive TrkB neurons was decreased sharply in 2d group after hSCI, and gradually increased and reached a peak level in 30d group. But in 60d group the number decreased again. In 90d group the number showed a continually decreasing trend. (4) In addition, there was no significant difference (p>0.05) in the number of the BDNF and TrkB immunoreactive neurons of the ventral horn between the operation side and the intact side of the thoracic cord, only except 30d group in BDNF and 2d and 14d group in TrkB.Conclusion: 1. In the adult rhesus monkeys in sham-operational control group and all SCI groups, the positive BDNF and TrkB immunoreactive neurons were observed in the ventral horn of the thoracic cord gray matter. This suggests that BDNF and TrkB may participate in the physiological function of the ventral horn neurons of the spinal cord .2. After hSCI, the animals showed a spontaneous functional recovery. During this period of time, there was a similar change in the expressions of BDNF and TrkB in the ventral horn of the thoracic cord comparing with the sham-operation control group. This indicates the expressions of BDNF and TrkB may be involved in and improve the regeneration of the neurons, and enhance the recovery of the spinal cord after injury.3. A very close trend of the expression change in both BDNF and TrkB the ventral horns after hSCI was found at the late stage after hSCI in the present experiment. That was the number of the positive immunoreactive neurons increased sharply and kept increasing to a peak level, and then continually decreased. This suggests that BDNF has a concordant function with TrkB, and it needs a specific mediation of its high affinity receptor TrkB to exert an important effect in maintaining neurons alive and even rescuing injured neurons. This tendency of expression changes of BDNF and TrkB we found in this study may provide a theoretical evidence for the clinic treatment to SCI.4. The number of BDNF and TrkB immunoreactive neurons decreased sharply at the earlier stage after SCI. It suggests that the optimal therapeutic window should be in the early stage following SCI.
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