Research On PD-L1 Expression In Contusion And Laceration Of Brain

BackgroundTraumatic brain injury(TBI)can cause a high disability rate,and induce a huge burden on many families and society.The pathological process of TBI includes: primary injury and secondary injury.Secondary injury is the key pathological process after TBI,which impact the prognosis.There are many mechanisams associated with secondary injury,in which inflammatory response after TBI plays a significant role in the treatment and its prognosis.In recent years,many studies pay attention into PD-1/PD-L1 pathway which plays an important role in immune regulation.PD-1/PD-L1 signalling pathway can regulate the activity of macrophages and lymphocytes cells and their cytokines secretion.There are many researches about PD-1/PD-L1 signalling pathway in the areas of tumor,autoimmune diseases and infections,while research on traumatic dieases is rare.PD-L1 expression in inflammatory cells after TBI has not been explored,so we designed this experiment to solve this problem.Objective Experiment 1:As microglia is the most important cells for innate immunity in CNS,we designed blast injury model by using dogs.Brain tissues were collect at different times after blast injury.Immunohistochemistry methods were used to explore the expression features of PD-L1 in microglia of brain tissues after blast injury.Experiment 2:We did immunohistochemistry and H-E staining to explore the number of inflammatory cells in specimens after traumatic brain injury at different time points.The characteristic of PD-L1 expression on the microglia,astrocytes and neurons in the contusion and laceration of brain after TBI were also detected.In a word,this experiment may provide new therapy by intervening PD-1/PD-L1 signalling pathway.Methods Experiment 1:28 female beagle dogs were used to make the blast injury model.The beagle dogs were 8-10 kg.The brain tissues were collect instantly when the dogs were died after blast injury and immersed and fixed by using 4% paraformaldehyde at 4℃.And there were five groups: normal group(5cases),30 min group(7cases),2h group(6cases),5h group(5cases),and 2d group(5cases).Frozen sections were made by using Leica freezing microtome.Then PD-L1 and Iba-1 immunofluoresent staining were performed and 4 area of interest were choose to detect the positive cell numbers of microglial cells expressing PD-L1 by using Image pro plus 6.0 software.Experiment 2:Between the year of 2014 and 2015 in the people’s liberation army(PLA)153th central hospital,we collected contusion and laceration of human brain tissue from the patients with TBI.The number of contusion and laceration of human brain tissues is 15;and the tissues were divided into 6h group(5cases),12 h group(4cases),1d group(3cases),4d group(4cases)and the normal group(7cases).All the specimens were fixed by using 4% paraformaldehyde.H-E and immunofluoresent staining were carried out in Frozen sections.The number of infiltrated inflammatory cells was detected.And microglia,astrocytes and neurons expressing PD-L1 were analysed.The specimen collection and use of brain tissues were under the supervision and agreement of the hospital ethics commitee.Patients’ families had been signed on papers.Rusults Experiment 1:The expression of PD-L1 in microglial cells started to increase at 5h time point(P < 0.01),and obviously increased at 2d time point(P < 0.05).Experiment 2:1.We detected the inflammatory cells in frozen sections by using H-E staining.The results shows: after contusion and laceration of human brain,the number of infiltrated inflammatory cells didn’t have any changes,and the infiltrated inflammatory cells increased significantly at 1 d and 4d time points(P < 0.01).2.After contusion and laceration of human brain,microglia cells were activated.And we detected the positive cells numbers of expressing PD-L1,Iba-1 and Hoechst33342.The results shows that: the positive cells started to increase at 6 h(P < 0.01),and increased significantly at 12 h,1d,and 4 d after injuyr(P < 0.05).3.Neurons and astrocytes were also could express PD-L1 after contusion and laceration of human brain.Conclusion1.The inflammatory paticipates in secondary injury after contusion andlaceration of human brain.2.PD-1/PD-L1 signalling pathway participates in the inflammatory of secondary injury,Intervation the PD-1/PD-L1 signalling pathway may be a new method to cure the cerebral contusion and laceration.