| Bachground:Spinal injury is a serve trauma. For the reason of its conventional therapeutic efficacy not well, a lot of patients remained sequela such as dyskinesia, sensory disability, dysfunction of bladder and rectum. Neurotrophic factor is essential material for nerve injury recovery and functional reconstruction.To begin with correlated molecule mechanism of endogenous NT-3 and its receptor TrkC expression,,exploring new strategy to promote spinal injury recovery is one of significance content about international neuroscience researching area.Objective:To investigate the phase changes of NT-3 and TrkC from relative motor neurons in injury segment bilateralis of adult Sprague-Dawley rats subjected to cord transection by both immunohistochemistry, Western blotting and RT-PCR, and also give primary discussion about the temporal changes and endogenous action mechanisms of endogenous NT-3 and TrkC following transected spinal cord injury,and make understanding its relationship with plasticity of spinal cord.Method: Adult female Sprague-Dawley rats (200g-220g) were used in our study. Adult SD rats were divided into six groups randomly with seven rats each group.One group is sham operation and the other groups for spinal cord transaction. Spinal cord transection were performed at T9 to T10 level in rats from five groups, while control group did not receive transection of the spinal cord. The rats received spinal cord transection were allowed to survive for 1 days,3 days,7 days,14days and 28days.While the time point reach,experimental rats were sacrificed. Seven tissue sample were removed such as two sides cerebral cortex, midbrain,4 columns from caudal and rostral ambi-1 cm spinal cord around the injury site corresponding T8 to T11 level. After perfusion, Samples from 3 rats each group were processed for NT-3 and TrkC immunohistochemistry. And then, fresh sample from 2 rats each group were obtained for the use of sem-qualification by western blotting and qualification by RT-PCR. Hindlimb function was evaluated and recorded after operation. Through postfix and dehydration,The sample for immunohistochemistry was cut to 20μm thick frozen sections. By the irnrnunohistochemical SP and fluorescence staining method, we observed the morphology,distribution and subcellular localization of NT-3 and TrkC immununoreactive cells in the ventral horn of the spinal cord and the motor area of the cerebrum. The immunoreactive cells were counted and the Optical densities (OD) were measured. The protein of NT-3 and TrkC was measured by western blotting method for sem-qualification. The mRNA of NT-3 and TrkC was measured by RT-PCR for qualification.The data were statistically anaiysed with one-way ANOVA and LSD test using SPSS 13.0 statistical package.Result:1. Western blotting strap specificity recogniting molecular mass 13.6Kda and 145Kda corresponded molecular mass of NT-3 and TrkC respectively. Replace experiment and negative control experiment is negative. 2.According to the irnrnunohistochemical outcome, Varying intensities of NT-3 and TrkC immnuostaining were observed in a wide variety of motor neurons, endaxoneuron and glial cells in the rat central nervous system. Through sem-qualification by western blotting, The most high concentration of NT-3 was detected in brainstem and hippocampus of rat brains.3. The dense NT-3 and TrkC positive products can be observed in the cytoplasm and processes of neurons in sham group and experimental groups.The nuclei and cytoplasm were labeled by NT-3 antibodies.Robust labeling of NT-3 were detected in nuclei. The dense TrkC positive products can be observed in the cytoplasm of neurons, but very few in the processes and nerve terminal, not be observed in the motor cortex.After spinal cord injury, the nuclei stain intensificated obviously and also been observed in some arborescence branch and neurite,but not be observed in centre motor cortex of brain.4. (1)The means of NT-3 positive neurons from caudal and rostral spinal cord around the injury site have significant deviation (P<0.001). The means of NT-3 positive neurons from cornu anterius medullae spinalis in each experimental group have significant deviation versus sham group (P<0.05). Positive neurons with NT-3 were sharply decreased in 1ST day after SCI. After 1ST day, the number of the NT-3 positive neurons were constantly increasing, at the end of 28th,maintaining the increase tendency. The means of NT-3 positive neurons from caudal and rostral spinal cord around the injury site in each group have no significant deviation (P=0.184-1.000). (2) The means of TrkC positive neurons from caudal and rostral spinal cord around the injury site have shown down regulation versus sham group in 1ST day and 7ST day after SCI, and then,have recovered to the level of sham group in 14ST day and 28ST day after SCI.The expression level of TrkC from rostral was higher than caudal significantly. The expression level of TrkC from injury segment was higher than consecutive segment significantly. Sem-qualification by western blotting can not detected TrkC protein in motor cortex of brain. (3)Resembling to the changes of TrkC protein, mRNA level of TrkC was decreased significantly from 1ST day to 7ST day after SCI versus sham group, but recovered to super-level of sham group in 14ST day.Contrasting sham group, The expression of TrkC mRNA remained a lower level in motor cortex of brain at different time point. (4) The OD of NT-3 and TrkC in cornu anterius medullae spinalis had been a similar tendency with the changes of irnrnunohistochemistry after SCI.5.Immunoreactivities of NT-3 positive neurons in the fore motor area of cerebral cortex had been a similar tendency with the changes of the motoneurons in the ventral horn after SCI.6. The BBB score of the sham-operated and normal rats were 21, after spinal cord transection, the rats showed complete paralysis (0), however, spontaneous functional recovery of the rats'hindlimbs occurred during the time-course, and peaked at 28d (2.57).Conclusion:1. Our antibodies identification experiments showed that the antisera were specific for the appropriate neurotrophin and did not cross react with other neurotrophins.2. NT-3 and TrkC were distributed in motor neurons in limited regions of adult brains of rats. NT-3 likewise its receptor TrkC, in majority of regions of rodents brains and ventral horn, having the overlapping expression, but differences also exits,and so did the expression of TrkC. All above indicated NT-3 and TrkC participate the physiological function of nerve cells in the brain and spinal cord of rodents, nevertheless, they performed different action respectively in different region. 3. The temporal changes differences between the positive neurons with NT-3 and that with TrkC after SCI, indicating the expression of NT-3 and TrkC have the relation to the recovery after SCI. However, some discrepancies also can be observed for their mode of action in different time course after SCI. It can offer some theory based on experiment for clinic using NTFs to cure spinal cord injury. |
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