The Expression Changes Of Mfn2and GFAP After Spinal Cord Injury And Astrocytes Scratch Cultured In Vitro

Objectives: To explore the expression changes of Mfn2(mitofusions-2, Mfn2) and GFAP(glial fibrillary acidic protein, GFAP) after spinal cord injury and in vitro astrocytes scratchand analysis its significance, which provide a basis for the next research aboutoverexpression of Mfn2suppressing the astrocytes activation after SCI (spinal cord injury,SCI).Methods: To build the rat model of spinal cord injury and the scratch model of astrocytescultured in vitro, then to detect the expression of Mfn2and GFAP using the methods ofimmunofluorescence-staining and Western Blotting. Part1:48adult male SD rats wererandomly divided into two groups: sham group (sham group, n=24) and spinal cord injurygroup (SCI group, n=24). The animal of SCI group were made thoracic spinal cordinjured model according to the Allen’s method but the animal of sham group were onlymade the operation of laminectomy. Animal were anesthetized and the thoracic spinal cordwas taken at the day of1,3,7and14separately. Then his tic-immunofluorescence-stainingwas made to observe the morphology of the spinal cord and the Western Blotting was madeto detect the expression changes of GFAP and Mfn2at various points in time. Part2: Theprimary cultured astrocytes were purified and then sub-cultured in culture dish of3.5mmdiameter (each group was repeated two times), then the astrocytes were randomly divided into five groups: control group (n=6),1-hour group (n=6),12-hour group (n=6),24-hourgroup (n=6) and48-hour group (n=6). When the astrocytes were grown up to80-90%alignment, the astrocytes were scratched by sterile lines according to the shape of “#”, butthe control group didn’t do any process. Cell immunofluorescence staining was made toobserve the morphology change and the Western Blotting was made to test the expressionchanges of GFAP and Mfn2at the1-hour,12-hour,24-hour,48-hour after scratched.Result：①The immunofluorescence staining result showed that after spinal cord injured,the thoracic segment between dorsal horn of spinal cord grey matter was damaged and thestructure was disordered and the boundary was not clear; the result of Western Blotting wasshowed that after spinal cord injury, the expression of Mfn2in the thoracic spinal cord wasdecreased from the first day, compared with sham group, the Mfn2expression of group ofthoracic spinal cord injury was decreased, and from the day of3after operation, theexpression of Mfn2were decreased obviously, the differences were statistically significant(p＜0.05), the expression of GFAP was increased gradually with the development of injury,the differences were statistically significant from the day of3after the operation (p＜0.05).②Immunofluorescence staining result found that after the astrocytes cultured in vitrowere scratched, the astrocytes were activated, the body of cells were increased, theprojections were also increased, and the expression of Mfn2was decreased; the result ofWestern Blotting found that compared with control group, the group of astrocytes werescratched, the expression of Mfn2were decreased, the differences were statisticallysignificant (p＜0.05) from the time of12-hour, however, the expression of GFAP wasincreased, the differences were statistically significant (p＜0.05) from the time of24-hour.Conclusion：①After the injured of thoracic spinal cord and the scratch of astrocytescultured in vitro, the expression of GFAP which is the specific marker of astrocytes wasincreased, this is pointed out that the astrocytes was activated and proliferated after the model of spinal cord injury and the scratch; At the same time, after the injured of thoracicspinal cord and the scratch of astrocytes cultured in vitro, the products of Mfn2which isproved to be an inhibited gene was decreased. So there is an inversed relationship betweenthe expression of Mfn2and the activation and proliferation of astrocytes after the injured ofthoracic spinal cord and the scratch of astrocytes cultured in vitro.②We first demonstratedthe relationship between the endogenous Mfn2’s expression decrease and the astrocytesover activation, which is provided foundation for the further research of the mechanism.