Alteration Of Microglia Function By Granulocyte-colony Stimulating Factor In Spinal Cord Injury Model Mice

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor, which was secreted by monocyte-macrophage, fibroblast and endothelial cells. They can combine with the specific cell surface receptor G-CSFR (CD114) to promote neutrophil progenitor cell growth and differentiation. G-CSF protects neutrophils from apoptosis and enhances their function. Over the past decade, the G-CSF was used to mobile hematopoietic stem/progenitor cells and mesenchymal stem cells of bone marrow donor to the recipient peripheral blood and realized peripheral blood stem cell transplantation. G-CSF has become the effective drugs of prevention and treatment with a variety of neutropenia. The resent study found that G-CSF has a protective effect of the central nervous system through the blood-brain barrier. The study greatly expands the clinical application of G-CSF.G-CSF was demonstrated a significant neuroprotective effect of cerebral ischemia in mice. G-CSF can reduce the infarct volume which was induced by glutamate excitotoxicity and improve neuron survival. G-CSF can cause the formation of new nerves and blood vessels within the damaged area, weaken the tissue damage, reduce infarct volume and improve neurological function. Our laboratory group demonstrated that G-CSF mobilized neural stem cells in mice to treat with hypoxic-ischemic brain injury. This study found that G-CSF reduced neuronal apoptosis by regulating protein, and the G-CSF can promote the SVZ and DG such as region-derived NSCs proliferation, which may provide new ideas for the clinical application of G-CSF treatment of nerve injury-related diseases.Spinal cord injury is a severe hazard to life and health disease, a lot of studies on spinal cord injury. Microglia (MG), as representative of the immune cells in the nervous system,are widely distributed in the central nervous system. They play an important role in nutrition, protection and restoration of neurons. MG are like a "double-edged sword" in the nervous system damage, diseases and other pathological conditions. They have different morphologies and functions, and have a variety of immune activities, can play the dual role of protection and destruction. Gordon S divided macrophage into two types according to activation signaling pathways: classically-activated (M1) and alternatively-activated (M2). Two types of cells have different immunological phenotypes and functions. M1cells secrete different cytokines to promote inflammation, inhibit tumor formation. M2cells have active phagocytic function, suppress inflammation, promote tissue regeneration and repair. Therefore, there are great significances to study the protective effect of MG of spinal cord injury, inhibition of the damage effect on the repair of spinal cord injury. This study observed the conversion of G-CSF on the dual role of microglia: M1/M2microglia in spinal cord injury, and investigated the possible mechanism of G-CSF on microglia.The first part:the effect of G-CSF on the motor functional recovery and distribution of microglia after mice spinal cord in vivoFirst we established the right of the mouse spinal cord hemisection injury model. Then model was given granulocyte colony-stimulating factor for3consecutive days, Basso-Beattie-Bresnahan (BBB) score to evaluate the motor function of mice. Spinal cord slices were immunofluorescence stained to raise the number and morphology of microglia and the different distribution of microglia within the gray and white matter. BBB score indictated that G-CSF improved mice locomotor recovery after spinal cord injury. And more IBA1+cells were recruited to the injury site and the surrounding in the mice receiving G-CSF treatment compared with injury group.The second part:the effect of G-CSF on microglia cell line under the conditions of the spinal cord injury in vitroImmunofluorescence staining and RT-PCR technique were used to detect the expression of G-CSFR in BV2glial cell membrane. MTT assay was used to evaluate the effect of G-CSF on the microglia activity. M1/M2microglia-related gene expression and cytokine mRNA levels were detected in the injured spinal cord tissue supernatant simulation environment of the spinal cord injury in vitro. Western blotting analysis the changes of microglia NF кB p65signaling pathways activity and STAT3, p-STAT3expression level after G-CSF treatment. We concluded that administration of G-CSF for the first72hours after spinal cord injury might reduce early inflammation-induced detrimental effect and promote an anti-inflammatory response that favors repair. Therefore, administration of G-CSF in the acute phase of spinal cord injury may be a promising strategy in restorative therapy after spinal cord injury.In conclusion, we have demonstrated G-CSF can regulate microglia activation in vivo and promote recovery of motor function, and it can also alter microglia polarization process and cytokine secretion to play a neuroprotective role.