Effect And Mechanism Of FTY720 On Blood-Brain Barrier After Traumatic Brain Injury

Objective: Traumatic brain injury(TBI)is one of the major health problems in the world.It is the main cause of death and long-term disability in patients with brain injury.After TBI,the damaged brain tissue initiates the cascade reaction of inflammation and oxidative stress,leading to secondary brain injury,which further leads to brain edema,hemorrhage,ischemia and other pathophysiological changes to damage the normal brain tissue.In this study,the effects and potential mechanisms of FTY720,a drug approved by FDA,on blood-brain barrier(BBB)after TBI were explored.The effects of FTY720 on inflammatory response,neurological function and survival after TBI were also evaluated.It is hoped that this study can provide valuable basic research data and guidance for the treatment of clinical TBI patients.Methods:1.Standard experimental male ICR mice were fed,and the TBI model was established by the weight-drop method.2.The injured brain tissues at different time points after TBI were collected to perform Western blot to detect the expression levels of S1pr1,Claudin-5 and Occludin.3.The selected time point for the subsequent experiment was determined by the time-course of protein expressions which were measured in the previous experiment.After determining the time points for subsequent experiments,the mice were randomly divided into 4 groups: Sham operation group(Sham),TBI group(TBI),Vehicle control group(TBI + Vehicle),and drug intervention group(TBI + FTY720).Mice in the Sham group were sutured immediately after scalp incision;mice in the TBI group were induced TBI after scalp incision;mice in the Vehicle control group were intraperitoneally injected with normal saline after TBI induction;mice in the drug intervention group were intraperitoneally injected with FTY720 after TBI induction.4.The grouping is the same as in the previous step.Proteins of ERK pathway and tight junction proteins were evaluated by Western blot and immunofluorescence.BBB integrity and brain water content were evaluated by Evans blue and dry-wet weight method respectively.5.GFAP was used to label astrocytes,IBA-1 was used to label microglia,and immunohistochemistry was used to label the activation of two kinds of cells in the contusion brain after TBI.6.The neurological function of mice in each group was evaluated by the m NSS.Finally,the mortality rates of mice in all groups except the Sham group were analyzed.Results:1.The expression of S1pr1 in the injured brain increased over time after TBI(compared with the Sham group,one day after TBI p < 0.05,three days after TBI p < 0.001),while the expression levels of the two tight junction proteins decreased and reached the lowest level on the first day after TBI(compared with the Sham group,p < 0.01 for Claudin-5,p< 0.05 for Occludin).2.After the administration of FTY720,the number of apoptotic of endothelial cells in mice after TBI was decreased compared with that in the TBI group and the TBI + Vehicle group(p < 0.001).3.After administration of FTY720,the expression of tight junction proteins were up-regulated(compared with the control group and the Vehicle control group,p < 0.05 for Claudin-5,p < 0.05 for Occludin),and the ERK pathway was inhibited(p < 0.05),the exudation of Evans blue was reduced(p < 0.001)and the brain water content was also decreased(p < 0.05).4.FTY720 attenuated the activation of astrocytes(p < 0.01)and microglia(p < 0.01).5.FTY720 improved neurological function on the first day after injury in TBI mice(p <0.01)and significantly increased the survival rate of TBI mice(p < 0.05).Conclusion: The purpose of this study was to investigate the function and status of endothelial cells after TBI.FTY720 inhibited the apoptosis of endothelial cells,improved the damaged BBB,and improved the neurological function after TBI.In addition,FTY720 attenuated the activation of astrocytes and microglia and reduced the inflammatory response in brain tissues after TBI.These results suggest that FTY720 therapy can promote the recovery of BBB and neurovascular unit(NVU)after TBI.This study also confirms that S1pr1 and ERK related signaling pathways may be the potential targets for the treatment of TBI.