Protective Effects Of Mitochondrial Division Inhibitor-1 And Its Mechanism In Rats With Acute Spinal Cord Injury

ObjectiveTo detect the effects of the mitochondrial division inhibitor-1 (Mdivi-1) on the mitochondrial injury and apoptosis after (acute spinal cord injury, ASCI) in rats. This study contributes more insight into the mechanism of spinal cord secondary injury, and provides a new way of preventing and treating ASCI.MethodsSprague-Dawley rats were randomly divided into four groups:(1) Sham-operated group (Sham group), (2) Sham+Mdivi-1 pretreatment group (1.2 mg/kg, Sham+Mdivi-1 group), (3) spinal cord injury group (SCI group), and (4) SCI and Mdivi-1 pretreatment group (1.20 mg/kg, SCI+ Mdivi-1 group). The rats in the SCI and SCI+Mdivi-1 groups were underwent a injury at thoracic level 10, using an established Allen's model. In the Sham+Mdivi-1 and SCI+Mdivi-1 group, the rats were given Mdivi-115 min before laminectomy or ASCI through an intravenous bolus. The rats in SCI group were sacrificed at 2,4,8,16, and 24 hours (h) after ASCI. The rats in the Sham, Sham+ Mdivi-1, and SCI+Mdivi-1 groups were sacrificed at 16 h after laminectomy or injury. Firstly, We examined the time-dependent change in the release of Cytochrome C (Cyt C) in mitochondria, then chose to detect the effect of Mdivi-1 at 16 h after ASCI. Next, mitochondrial morphology was detected by transmission electron microscopy. Mitochondrial membrane potential of rats in each group was deteced by JC-1 labeling. Mitochondrial adenosine triphosphate (ATP), malondialdehyde (MDA), and reduced glutathione (GSH) of rats in each group was measured with a VIS spectrophotometer. The content of Dynamin-related protein1 (Drp1),Cyt C, Bcl-2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl-2) on the mitochondria, and Caspase-3 protein in cells in each group were detected by western blot. The expression level of caspase-3 of spinal cord tissue in rats were measured by immunofluorescence technique. The number of apoptotic cells of rats in each group were assessed by TUNEL staining.Results1. Western blots revealed that in comparison with the Sham group, the protein levels of Cyt C on mitochondria decreased significantly at 4 h, and reached a minimum level at 16 h in the SCI group, and increased significantly from 16 to 24 h (P< 0.01). While, the protein levels of Cyt C in the cytoplasm increased significantly at 4 h and reached a maximum level at 16 h in the SCI group, and decreased significantly from 16 to 24 h (P< 0.01). Compared with the Sham group, the expression of Drp1, Bax and Bcl-2 on mitochondria and total caspase-3 were significantly increased in the SCI group at 16 h (P< 0.01). Compared with the SCI group at 16 h, the level of Cyt C in the cytoplasm, Drp1 and Bax on mitochondria, and total active caspase-3 were significantly decreased in the SCI+Mdivi-1 group (P< 0.01), and the level of Cyt C in the mitochondria were significantly increased in the SCI+Mdivi-1 group (P< 0.01), but the level of Bcl-2 on the mitochondrial outer membrane did not change. The basal levels of the above indicators did not differ in the Sham group and in the Sham+Mdivi-1 group.2. Transmission electron microscopy results showed that compared with the Sham group, the absolute numbers of mitochondria increased significantly and the individual mitochondrial cross-sectional area decreased significantly in the SCI group at 16 h (P< 0.01). compared with the SCI group at 16 h, the absolute numbers of mitochondria reduced significantly and the individual mitochondrial cross-sectional area increased significantly in the SCI+Mdivi-1 group (P< 0.01). However, the basal levels of the mitochondrial morphology did not differ in the Sham group and in the Sham+Mdivi-1 group.3. JC-1 labeling showed that compared with the Sham group, the mitochondrial membrane potential decreased significantly in the SCI group at 16h (P< 0.01); compared with the SCI group at 16 h, the mitochondrial membrane potential were maintained significantly in the SCI+ Mdivi-1 group (P< 0.01). However, the basal levels of the mitochondrial membrane potential did not differ in the Sham group and in the Sham+Mdivi-1 group.4. VIS Spectrophotometer showed that compared with the Sham group, the mitochondrial ATP and reduced GSH levels decreased significantly, while MDA level increased significantly in the SCI group at 16h (P< 0.01); compared with the SCI group at 16 h, the mitochondrial ATP levels were maintained significantly, MDA levels decreased significantly, but reduced GSH levels increased significantly in the SCI+Mdivi-1 group (P< 0.01). However, the basal levels of mitochondrial ATP, reduced GSH.and MDA level did not differ in the Sham group and in the Sham+Mdivi-1 group.5. Immunofluorescence results showed that compared with the Sham group, the percentage of active Caspase-3-positive cells increased significantly in the SCI group at 16 h (P<0.01); compared with the SCI group at 16 h, the percentage of active Caspase-3-positive cells decreased significantly in the SCI+Mdivi-1 group (P< 0.01). The basal levels of the percentage of active Caspase-3-positive cells did not differ in the Sham group and in the Sham+Mdivi-1 group.6. Fluorescence TUNEL assay showed that compared with the Sham group, the percentage of apoptotic cells increased significantly in the SCI group at 16 h (P< 0.01); compared with the SCI group at 16 h, the percentage of apoptotic cells decreased significantly in the SCI+Mdivi-1 group (P< 0.01). The basal levels of the percentage of apoptotic cells did not differ in the Sham group and in the Sham+Mdivi-1 group.ConclusionsMdivi-1 can significantly protect the mitochondrial membrane potential and ATP levels, inhibit the mitochondrial oxidative damage and the activation mitochondrial apoptosis pathway after acute spinal cord injury.