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Effects of FK506 on the mRNA and Protein Expression of Axon Guidance Cue Slit-2 after Spinal Cord Injury in Rats

Author: LiKaiFu
Tutor: LiuWenGe
School: Fujian Medical
Course: Surgery
Keywords: spinal cord injury FK506 nerve growth factor axon guidance cue slit-2
CLC: R651.2
Type: Master's thesis
Year: 2011
Downloads: 35
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Abstract

Objective To investigate the functional and pathology changes and the treatment effect of FK506 on the expression of axon guidance cue slit-2 after spinal cord injury in rats, thus providing reliable data for clinical therapy of spinal cord injury(SCI).Methods A total of 150 adult Sprague-Dawley rats were randomly divided into three groups, that is, the sham operation group (n=30), the injury group (n=60) and the FK506 treatment group (n=60). The T10 SCI model was injured by using the modified Allen’s weight dropping technique and the rats in last group were given FK506 (0.3mg/kg) by peritoneal injection after SCI. The sham operation group and the injury group were given the same amount of saline instead. All animals were evaluated of the hind limb behavior with Basso-beattie-bresnahan(BBB) score on five different time-point (1, 3, 7, 14 and 28d).After the BBB evaluation, the rats were sacrificed and the spinal cord was taken out. HE stain was done in order to observe the morphologic changes. The expression and significant of slit-2 were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical technique.Result (1) The result of BBB scales: In the sham group, the hind limb behavior function was normal, and the BBB score reaches 20 point, there were nerve functional disorder in the injury group and the FK506 treatment group, which BBB score less than 2 point at the day of 1and 7, and was no statistical difference between later groups(P>0.05). Following, the nerve functional of FK506 treatment group was much better than the injury group, and the score increasing with time, with a significantly statistical difference among each group. (2) HE stain result: The boundary between grey and white matter is clear, the structure of the neuron and the myelinic membrane is normal. But there were changes to certain degree between the injury group and treatment group, that is, some hemorrhagic foci, inflammatory cells infiltration, the cell bodies of neurons atrophy, the reactivity proliferation of neuoglia cells, the white matter appeared reticulate demyelination and glial scar formation. The result suggest that the treatment group better than the injury group. (3) The result of immunohistochemical examination of slit-2: The positive signal expression of slit-2 appeared a few scattered in anterior horns of spinal cord in the sham group, and it appeared obviously in gray matter and in neuronal cytoplasm in the in the injury group and the FK506 treatment group. The expression of slit-2 changed with time. The expression of slit-2 could be detected at day 1 after SCI, reached the highest at day 7 and then decreased gradually, with higher expression level in injury group compared with treatment group(P<0.05). And, there have significantly statistical difference at days point 3 and 7 after SCI of variance analysis in injury group compared with treatment group(P<0.01). (4) RT-PCR result: the expression of slit-2 mRNA was low and level off in the sham operation group, which changed with time between the injury group and treatment group. The expression of mRNA was observed at day 1 after SCI, and increased at day 7, reached peak on the 7th day, declining gradually later, with higher expression level in injury group compared with treatment group(P<0.05). And, there have significantly statistical difference at days point 3 and 7 after SCI of variance analysis in injury group compared with treatment group(P<0.01).Conclusion Low-dose FK506 could improved the motion function to a certain degree and up-regulated apparently the expression of axon guidance cue slit-2 , which may act as inhibitory role to repel anon regeneration via mediating migration of inflammatory cells and secretion of factors, slit-2 is likely to do something importantly with axon regeneration after SCI.

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