Microglia Promote Vascular Repair And Functional Recovery After Spinal Cord Injury Through Overexpressing BDNF

ObjectiveTo explore the molecular mechanism of overexpression of brain-derived neurotrophic factor on microglia to promote vascular repair after spinal cord injury and its protective effect on nerve and motor function in mice.MethodsIn this experiment,the spinal cord injury model was established by the spinal cord clamp injury method,and then the mice were divided into six groups according to the principle of random grouping according to the genetic requirements of each group of mice.Group（1）is a sham operation group（sham）: only the lamina of the T10 segment of the spinal cord was removed without damage,and the mice used in this group were C57 BL/6J;group（2）was a spinal cord clamp injury group（crush）: The mice were modeled with T10 spinal cord clamp injury,and the mice used in this group were C57 BL/6J;group（3）was the spinal cord clamp injury + PLX5622 deplet microglia group: the mice in this group were treated two weeks before modeling.The mice were given the diet containing PLX5622 at the beginning,and continued to be given the diet containing PLX5622 after the spinal cord injury model was established.The mice used in this group were C57 BL/6J;group（4）was the spinal cord clamp injury group: for mice Modeling of T10 spinal cord clamp injury,the mice used in this group are CX3CR1 cre ER^-/+::LSL-BDNF^-/+-td Tomato;group（5）is the spinal cord clamp injury group,the mice used in this group are TMEM119 cre ER^-/+::LSLBDNF^-/+-td Tomato;group（6）is spinal cord clamp injury + PLX73086 deplete peripheral macrophages group: the mice in this group were given CSF1 R inhibitor that could not cross the blood-brain barrier two weeks before model establishment.The diet containing the CSF1 R inhibitor PLX73086 was given continuously after the spinal cord injury model was established.The mice used in this group were CX3CR1 cre ER^-/+::LSL-BDNF^-/+-td Tomato.Immunofluorescence was used to detect the phenotype changes of microglial cells in mice on the third day after spinal cord injury,and the effect of deplete microglia on the pathological changes after spinal cord injury was detected by immunofluorescence on the 14 th day.The BMS score scale was used to evaluate the motor function recovery of the mice on the 1st,3rd,7th,14 th,21st,and 28 th days after the establishment of the model,and the footprint test and rotarod experiments were performed,and blood flow changes were detected by laser speckle flowmeter before,5 minutes after,and 28 days after spinal cord injury modeling,and histopathological changes were observed by immunofluorescence on the 28 th day after modeling.In addition,in order to transplant and replace the cells of overexpressing BDNF,we have improved the cell transplant and replacement methods developed by Professor Peng Bo of Fudan University and others,which are called Mr BMT-X Ray and Mr BMTBusulfan respectively.In the study of cell replacement method,the mice were divided into three groups according to the principle of random grouping.1:Conjoined symbiotic spinal cord injury group,the blood cells were labeled with green fluorescent β-actin GFP mice and C57 BL/6J wild-type mice by surgery.Mice were sutured together,using 8 β-actin GFP mice and 8 C57 BL/6J wild-type mice;2: Mr BMT-X Ray group（PLX5622 depleted spinal cord microglial cells plus X-ray radiation myeloablative transplantation group）,bone marrow cells were extracted from 4 CX3CR1 cre ER^-/+::LSL-BDNF^-/+-td Tomato mice and transplanted to 8 C57 BL/6J wild-type mice to establish a spinal cord injury model;3: Mr BMT-Busulfan group（PLX5622 depleted spinal cord microglial cells plus Busulfan myeloablative transplantation group）,transplanted bone marrow cells from 4 CX3CR1 +/GFP mice to 8 C57 BL/6J wild-type mice,and only observed cell transplantation replacement ratio,no spinal cord injury modeling processing.ResultsOur data show that microglia play a very important role in spinal cord injury,microglia depletion is not conducive to the recovery of motor function in mice after spinal cord injury,and continuous clearing of microglia in the spinal cord will aggravate the injury after spinal cord injury pathological manifestations.Overexpression of BDNF on microglia can promote functional recovery after spinal cord injury in mice,and overexpression of BDNF on microglia can regulate the phenotype transition of microglial cells after spinal cord injury to the antiinflammatory Arg1,and it can promote angiogenesis and increased blood flow in the spinal cord,promote axonal survival after spinal cord injury,and improve hindlimb motor function in mice.The improved cell transplantation and replacement method can realize high-efficiency transplantation and replacement of spinal cord microglia.ConclusionsThe results of the study proved that the continuous removal of microglia in the spinal cord is not conducive to the recovery of spinal cord injury,and the overexpression of BDNF on the microglia in mice is conducive to vascular repair and motor function recovery after spinal cord injury,providing neuroprotection.