| ?Objective? Spinal cord injury(SCI)is one of the most serious nervous system disorders characterized by high morbidity and disability.Unfortunately,few therapeutic methods can be used to restore the locomotor function of SCI patients.Pathological process of SCI can be divided into primary and secondary injuries according to the different mechanism.The primary injury is caused by the initial external force acting directly or indirectly on the spinal cord.The secondary injury is based on the primary injury,through a series of physiological and biochemical mechanisms,especially the inflammatory response,leading to the destructive lesions of the tissue itself,further deepening and expanding the degree of damage.The inflammatory response after SCI is complex.Studies have shown that the inflammatory response caused by SCI has the dual effects of nerve damage and neuroprotection.The activation of astrocytes in the acute phase of SCI helps to seal and isolate the necrotic cells and reconstruct the blood-brain barrier integrity.In the later stage of SCI,due to the formation of excessive glial scar,it hinders nerve regeneration the recovery of motor function.Therefore,it is necessary to use its two-phase effects in SCI repair,and suppress the inflammatory response to minimize its damage,while selectively exerting its repair potential.Metformin,a first-line drug for type-2 diabetes,and studies have shown that metformin can effectively inhibit inflammation.However,our pilot study showed that high dosage and early-stage treatment lead to increased mortality and limited locomotor function recovery.This study aims to explore the optimal period for the treatment of spinal cord injury and improve motor function by using metformin,so that it can balance the two-phase effect of inflammatory reaction after injury,and provide a new clinical strategy for the treatment of SCI.?Methods? In this experiment,C57BL/6 mice with a body weight of 30 grams at 16 weeks old were used to establish a mouse model of spinal cord injury at T11.They were randomly divided into 3 groups: Control group,0dpi group and 3dpi group,10 in each group.The 0dpi group received 100 mg/kg metformin by intraperitoneal injection immediately after spinal cord injury for 7 days.The 3dpi group received 100 mg/kg metformin at 3 days after spinal cord injury and continued injection for 7 days,while the Control group used the same dose of PBS and was intraperitoneally injected after spinal cord injury for 7 days.BMS scores were performed at 1-7 days,14 days,21 days,35 days,and 42 days after spinal cord injury to observe the recovery of motor function;14 days,21 days,35 days and 42 days after spinal cord injury,PAS motor function test was performed on days;42 days after spinal cord injury,the spinal cord tissue of the mice was exposed for electrophysiological testing to observe the recovery of spinal cord signaling function;the spinal cord tissue was obtained for frozen section,and the spinal cord tissue of each group was obtained.Immunofluorescence staining of neuronal nuclear antigen(NeuN)was obtained to observe the preservation and regeneration of neurons under laser scanning confocal microscope;immunofluorescence of Glial fibrillary acidic protein(GFAP)was obtained to observe the glial scar formation in the spinal cord injury area.The activation of microglia and macrophages was observed by immunofluorescence staining with anti-ionized calcium binding adaptor protein(Iba-1)staining;Ly-6G/Ly-6C(Gr-1)Fluorescence staining was used to observe the accumulation of neutrophils in the lesion center after injury,reflecting the inflammatory changes in the lesion center.The expression of TNF?,IL-1?,IL-6 and IL-10 in the injury center was detected by Western blot.?Results? The survival rate curves were plotted according to the postoperative and post-treatment mortality of the experimental mice.The mortality rate of the 3dpi treatment group was 20% after 42 days,the mortality rate of the 0dpi treatment group was 70%,and the mortality rate of the Control group was 50%.Mortality statistics for one week after surgery showed a 20% mortality rate in the 3dpi and 0dpi treatment groups within the week,compared with 40% in the Control group.The BMS motor function score showed that the subjects of 3dpi treatment group and 0dpi treatment group have a clear and significantly higher BMS evaluation compared with those in the Control group from 14 days after operation,and the recovery of motor function in Sub-Acute treatment group was significantly better than that of Acute treatment group(P<0.01).By 42 days after surgery,the motor function scores of the Sub-Acute treatment group recovered to 6-8 points,which was significantly better than the Acute treatment group and the control group(P<0.05).The PAS motor function test showed that the rearing and walking function of the Sub-Acute treatment group were significantly better than the Acute treatment group and the control group(P<0.05).NeuN and GFAP immunofluorescence staining was performed on the injured areas of spinal cord tissue of each group.Laser scanning confocal microscopy showed that the nerve tissue of the Sub-Acute treatment group retained more nerves than the Acute treatment group and the control group.GFAP-positive cells were significantly reduced(P<0.05),and the area of lesions in the Sub-Acute treatment group was significantly smaller than that in the Acute treatment group and the control group.Iba-1 and Ly-6G/Ly-6C(Gr-1)immunofluorescence staining were performed on the injured areas of spinal cord tissue of each group.Laser scanning confocal microscopy showed that the Sub-Acute treatment group had smaller number of glial cells.The number of phagocytes was significantly reduced compared to the Acute treatment group and the control group(P < 0.01).There are more neutrophils were observed in the epicenters of 3dpi treatment group when comparing with Control group(P < 0.05),but no statistical difference in the number of neutrophils between 3dpi treatment group and 0dpi treatment group,and between Control group and 0dpi group.Electrophysiological tests of spinal cord tissue showed that the cortical potential response of the spinal cord of the control group was significantly lost after stimulation,and the cortical potential response induced by stimulation in the Sub-Acute treatment group and the Acute treatment group was significantly increased and the potential level of the Sub-Acute treatment group was higher than that of Acute treatment group.Western blot results showed that the expression levels of pro-inflammatory factors TNF?,IL-1? and IL-6 were significantly decreased in the Sub-Acute treatment group(P<0.05),while the expression of the anti-inflammatory factor IL-10 in the Sub-Acute treatment group was observed.The amount was significantly higher than the Acute treatment group and the control group.?Conclusions? 1.High-dose metformin treatment immediately after spinal cord injury can lead to higher mortality and delayed recovery of motor function;2.In the subacute phase of spinal cord injury,treatment with appropriate amount of metformin can inhibit neuronal apoptosis,reduce inflammation and reduce neutrophil aggregation,inhibit excessive proliferation of microglia and astrocytes at the injury site,thereby inhibiting the expansion of glial scar,thus promote repair of spinal cord injury;3.Treatment with metformin at subacute phase of spinal cord injury can promote the recovery of motor function. |
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