Reactive Astrocytes Protect Tissue And Preserve Function After Traumatic Brain Injury In Ephrin-B2 Knock-out Mice

Traumatic brain injury (TBI) is the most common neurosurgical disease, which is a major cause of death and disability, especially in children and young adults. Recently, with the continuous development of neurosurgical treatments and medications, the mortality and disability have been decreased significantly, but the recovery of brain injury is still not satisfactory. There is a high incidence of severe neurological disabilities, such as altered memory and/or personality, and partial or complete paralysis, which affect seriously the quality of patient's life. It is a long-time goal for neuroscientist how to reduce disability rate and improve the cure rate. More and more attentions have been focused on secondary brain damage after primary injury. Ephrin are cell-surface-tethered ligands for Eph receptors (Erythropoietin-Producing Hepatocellular Receptor), the largest family of receptor tyrosine kinases. Ephrin-B2 is a newly elucidated member of Ephrin family. During development, it plays pivotal roles in the development of nervous system, for example, axon guidance and angiogenesis. During adulthood, it also influences cell behavior and tissue plasticity, such as migration and boundary shaping,especially in repair of brain injury.Objective: To breed conditional deletion (or knock-out) of Ephrin-B2 (Ephrin-B2 KO) from astrocytes in GFAP-Cre-loxP mice, to establish traumatic brain injury model, and to explore expression of Ephrin-B2 gene and effect on repair after traumatic brain injury in mice.Method:1 To breed Ephrin-B2 KO mice with EphrinB2-Lox mice and GFAP-Cre mice, test the efficiency of the conditional gene deletion for off-springs. 2 Whether the ephrin-B2 KO mice were healthy: Check the size, weight, as well as the cytoarchitecture of brains, and evaluate the appearance of Ephrin-B2 KO astrocytes, including size, number and density.3 120 male, aged 10-12 weeks, 25-30g, healthy C57BL/6 mice and Ephrin-B2 KO mice were used individually. Traumatic brain injury (TBI) model was induced by using Controlled Cortical Impact Device.4 The animals were divided randomly into four groups, according to time points, including 3days, 7days, 14days and 21days. The wild type mice of same number were used as control group at every time point. Uninjured control group was kept with the other groups. Immunofuorescence and western blotting were employed to determine alternations in Ephrin-B2 and GFAP expression, the double labeling was used to detect alternations in the co-expression of positive cell of GFAP and Ephrin-B2 by confocol microscope. GFAP mRNA level in injured cortex and ipsilateral hippocampus after TBI was tested. The volume of necrotic cavity was measured with Nissl Staining combined the image analysis system software. The motor of coordination and balance with Rotarod test and Beam walking test were conducted.5 Astrocyte scratch wound assay: The number of astrocytes entering the wound area was determined by counting the number of nuclei or GFAP expressing cells.Result:1 Normal appearance of Ephrin-B2 KO mice, CNS and astrocytes: The macroscopic appearance, size, and weight, as well as the cytoarchitecture of brains were indistinguishable from those of wild type littermates at all ages. Quantitative analysis of cells stained for GFAP indicated that all the cell size, the shape and the cell density of astrocytes did not differ significantly in brain of WT and KO mice. But western blotting revealed expression of GFAP in KO brain is a little higher than that in WT brain.2 Ephrin-B2 was strongly upregulated on the reactive astrocytes at the lesion epicenter in WT mice during 21days post-injury. Ephrin-B2 immunolabeling was primarily confined to cell bodies and the major process branches. There is no expression of Ephrin-B2 on the reactive astrocytes in KO mice.3 Expression of Ephrin-B2, pEphrin-B2 and GFAP are substantially increased after TBI in WT mice. Western blotting analysis also showed that, at 3 days after TBI, expression of Ephrin-B2 exhibited small attenuation, but not absent, then gradual upregulation. More important, there is the same trend of change to pEphrin-B2 and GFAP.4 Western blotting analysis also showed that Ephrin-B2 mice exhibited significant upregulation of GFAP compared with non-injured sham group and WT mice. It reached 4.7-fold for KO mice to WT mice at 3 days after injury, and attenuated to 2.8-fold. The same mRNA change was detected for GFAP at the lesion epicenter and ipsilateral hippocampus.5 Nissl staining showed Ephrin-B2 KO mice had smaller lesion volume at 21 days after TBI.6 Astrocytic behavior in a scratch wound assay revealed cell density was significantly greater in KO groups compared with WT groups either on 3 days or 7days after scratch. However, there was no significant difference between WT and KO mice in the size of astrocyte.7 Better recovery of motor functions after injury in KO mice.Conclusion: The present data detail the differential temporal and spatial expression profile of Ephrin-B2 after TBI. We find that Ephrin-B2 signaling exerts opposing effects on astrocytes reactivity. In Ephrin-B2 KO mice, the expression of GFAP and GFAP mRNA is up-regulated after injury, and astrocytes proliferate, migrate and become hypertrophic quickly and substantially, which improve the neuro-protection and decrease the secondary brain damage. Consequently, Ephrin-B2 KO mice have smaller lesion volumes. Thus, Ephrin-B2 KO mice enhance the functional outcome and decrease the sequelae after TBI. Therefore, when and how to adjust the signal transduction of Ephrin-B2, regulating the reactivity of astrocytes, reducing secondary tissue degeneration and augmenting their protective functions, can be a hope expected to an effective treatment of traumatic brain injury.