Glial Reactivity Following Diffuse Axonal Injury

Diffuse axonal injury(DAI)is caused by energy transmitted to the brain via rapid head rotation or deceleration/acceleration.Forces of tension,torsion and compression caused by different mass and movement brain tissue consequently lead to extensive white matter tract characterized by delayed axonal disconnection.Damage to myelin may be a major component of white matter injury and accompanied by complex reactions among glial populations.This study was to investigate the glial response and the relationship with secondary axonal degeneration in rats after DAI.Adult male Sprague-Dawley rats were randomly assigned to control or DAI groups sacrificed at 1,2,3,5 and 7 days with 10 rats in each group.DAI model was made referring to modified Marmarou method and the corpus callosum(CC)and brain stem were evaluated in the present study.We assess myelin loss using LFB staining and myelin basic protein(MBP)western blot(WB)analysis.Immunohistochemistry and WB were used to examine the glial expression of Olig2,Iba1,and GFAP.We evaluated the number of mature oligodendrocyte by CC-1 immunofluorescence staining and then further examined mature oligodendrocyte apoptosis by using double immunofluorescence staining for CC-1 and TUNEL.The ultrastructural changes of corpus callosum and brain stem were observed by transmission electron microscopy.The results of the experiments:(1)compared with the control,reduced LFB staining was observed at 3 and 7 days after DAI in corona radiata and brain stem,indicating myelin loss in DAI.There was a significant decrease in MBP expression at day 3 post DAI in CC compared to the control rats.While the MBP expression in brain stem was significant decreased in thefirst week after DAI,and the most obvious decreased was observed at 3days after DAI.(2)The oligodendrocyte expression of Olig2 in the CC of DAI rats increased significantly at 2d,3d and 5d after injury,while the expression of Olig2 in the brain stem was significantly increased compared with the control group within 1 week after DAI.(3)Compared with the control,the number of CC-1 positive mature oligodendrocytes in the CC and brain stem were significantly decreased at day 1 post DAI and it further decreased with the continuous extension of injury time.(4)Compared with the control,the number of CC-1/TUNEL positive apoptotic mature oligodendrocytes in the CC and brain stem were significantly increased at day 2 post DAI and it further increased with the continuous extension of injury time.(5)Compared with the control group,the number of Iba1-labeled microglia positive cells in the CC and brainstem of DAI rats was increased significantly,and different hypertrophic morphology of the Iba1-immunolabeled cells was identified after injury.Moreover,the WB results also showed increased expression of Iba1 from days 2 to 7 after injury in the CC and brain stem.(6)Following DAI,GFAP immunoreactivity showed marked staining of dense astrocyte processes within the CC.Quantification by WB analysis further confirmed that GFAP expression is significantly increased in the CC at 3,5 and 7 days post-DAI.However,there was no effect of DAI on GFAP expression in brain stem.(7)Transmission electron microscope results show that in control rats,the myelin sheath presented alternately dark and bright lamellae concentric configuration.The axolemma adhered tightly to the inner layer of myelin sheath and the axoplasmic contents such as microtubes,neurofilaments and mitochondria were distributed regularly.At 3 days after injury,the CC regions exhibit a focal disorganization of the myelin sheath and most axons contains reduced cytoskeleton and highly swollen mitochondria.Unlike CC,the brain stem regions exhibit widespread delamination of myelin lamellae at 3 days following DAI.The myelin extend outside or either inside.While,axonal cytoskeleton were generally organized and mitochondria appear intact.At day 7 after injury,both CC and brain stem showed obvious degenerating axons,manifested as cytoskeletal dissolution(light degeneration)or amorphous electron dense material(dark degenerion).Most of myelin lamellae were separated diffusely and disconnected locally.Moreover,myelin sheaths were collapsed around a degeneration axon and excessive myelin figures were observed.The above results indicate that mature oligodendrocytes have a selective susceptibility to DAI,accompanied by activation of microglia and astrocytes.Myelin loss caused by delayed oligodendrocyte death may be an important factor contributed to axonal secondary degeneration.Defining the cause of ongoing oligodendrocyte death and promoting myelin regeneration may be important targets for therapeutic interventions of DAI.