Spatiotemporal Characteristics And Mechanism Of Neuroblasts Migration After Traumatic Brain Injury

ObjectiveTraumatic brain injury(TBI)is a disease with high morbidity and mortality which leads to severe neurological dysfunction.However,there is still no effective treatment to solve this problem.Previous study showed that there are neural progenitor cells and neural stem cells in the subgranular zone(SGZ)and subventricular zone(SVZ)of the adult mammal’s brain,including human being.Interestingly,TBI not only causes brain damage,but also triggered neural migration.It is promising to explore the mechanism of neuroblasts migration after TBI for repairing damaged neurological functions.Brain-derived neurotrophic factor(BDNF),a member of neurotrophin family,is a secreted protein,which play a important role in the process of neural migration.However,the specific mechanism of BDNF in neuroblasts migration after TBI remains unclear.In present study,firstly,we established a mouse TBI model through Controlled Cortical Impact(CCI)to detect the spatiotemporal profile of neuroblasts migration,BDNF and reactive astrocytes derived BDNF.Secondly,we tested the effect of BDNF at different concentrations on migration of neuroblasts in SVZ explants.And then we investigated whether artificial intervention can induce neuroblasts migration in physiological mice and TBI mice by injecting different concentrations of TNF-α,IL-6,IL-1β or MCP-1.Moreover,we explored the fate of neuroblasts and cells in the target area induced by artificial intervention.Finally,we established a mouse TBI model through Controlled Cortical Impact(CCI)to detect the spatiotemporal profile of MCP-1.MethodsPart1 Spatiotemporal characteristics and mechanism of neuroblasts migration after traumatic brain injury.1.Thirty-six adult,male C57 BL / 6J mice were randomly divided into sham group(n = 6)and TBI group(n = 30),and then a moderate TBI model was established by CCI.Immunostaining was used to detect the spatiotemporal characteristics of neuroblasts migration on 1,3,7,14 and21 days after CCI.The morphological changes of neuroblasts in migration path on day 7 after CCI.2.Seventy-two adult,male C57 BL / 6J mice were randomly divided into sham group(n = 12)and TBI group(n = 60),and then a moderate TBI model was established by CCI.Immunofluorescent assay was used to detect the spatiotemporal characteristics of BDNF on 1,3,7,14 and 21 days after CCI.The level of BDNF in peri-SVZ and peri-lesion area were detected by enzyme-linked immunosorbent assay on day 1,3,7,14 and 21 post CCI.3.Thirty-six adult,male C57 BL / 6J mice were randomly divided into sham group(n = 6)and TBI group(n = 30),and then a moderate TBI model was established by CCI.Immunofluorescent assay was to demonstrate the cellar source of BDNF on day 7 after CCI.Immunostaining was used to detect the spatiotemporal characteristics of reactive astrocytes on 1,3,7,14 and 21 days after CCI.4.Thirty-six adult,male C57 BL / 6J mice were randomly divided into sham group(n = 6)and TBI group(n = 30),and then a moderate TBI model was established by CCI.Immunofluorescent assay was used to detect the spatiotemporal characteristics of reactive astrocytes-derived BDNF on 1,3,7,14 and 21 days after CCI.Immunostaining was used to explore spatial relationship between reactive astrocytes-derived BDNF and neuroblasts.5.Immunofluorescent assay was used to test the effect of BDNF at different concentrations(50ng/mL,100ng/mL and 500ng/mL)on neuroblasts migration in SVZ explants.Part2 Exploration of neuroblasts migration to be induced artificially.1.Three hundred and eighty-four adult,male C57 BL / 6J mice were randomly divided into control group(n = 24),MCP-1 group(n=72),TNF-αgroup(n=96),IL-6 group(n=96)and IL-1β group(n=96).The three intervention concentrations of MCP-1 were 50ng/mL,100ng/mL and500ng/mL.The four concentrations of TNF-α were 30ng/mL,100ng/mL,200ng/mL and 500ng/mL.The four intervention concentrations of IL-6were 40ng/mL,100ng/mL,200ng/mL and 500ng/mL.The four intervention concentrations of IL-1β were 20ng/mL,100ng/mL,200ng/mL and500ng/mL.Immunofluorescent assay was used to detect the characteristics of distribution of neuroblasts on 1,3,7 and 14 days after injection.Immunostaining was used to explore the characteristics of distribution of reactive astrocytes and reactive astrocytes derived BDNF on day 7 after injection.2.Forty-two adult,male C57 BL / 6J mice were randomly divided into control group(n = 6),MCP-1 group(n=18)and TNF-α group(n=18),and then a moderate TBI model was established by CCI.The three intervention concentrations of MCP-1 were 50ng/mL,100ng/mL and 500ng/mL.The three concentrations of TNF-α were 100ng/mL,200ng/mL and 500ng/mL.Immunofluorescent assay was used to detect the characteristics of distribution of neuroblasts,reactive astrocytes and reactive astrocytes derived BDNF on 7 days after injection in mice on day 28 post TBI.3.Eighteen adult,male C57 BL / 6J mice were randomly divided into MCP-1(50ng/mL)group,MCP-1(100ng/mL)group and MCP-1(500ng/mL)group(n=6).Immunofluorescent assay was used to explore the destiny of artificially-induced neuroblasts and cells in the target area on day14 after injection.4.Seventy-two adult,male C57 BL / 6J mice were randomly divided into sham group(n = 12)and TBI group(n = 60),and then a moderate TBI model was established by CCI.Immunostaining was used to detect the spatiotemporal characteristics of MCP-1 on 1,3,7,14 and 21 days after CCI.The level of MCP-1 in peri-lesion area were detected by enzyme-linked immunosorbent assay on day 1,3,7,14 and 21 post CCI.ResultsPart1 Spatiotemporal characteristics and mechanism of neuroblasts migration after traumatic brain injury.1.Neuroblasts migrated from SVZ to the lesion area,and arrived at the lesion area on day 7 after TBI.2.The level of BDNF was significantly higher in peri-lesion area than in peri-SVZ area on different time courses post-TBI,and this difference was most significant on day 7 after TBI.3.Neurons,reactive astrocytes,microglia and endothelial cells all contributed to BDNF expression after TBI.Reactive astrocytes might be a major factor stimulating BDNF expression after TBI.The number of reactive astrocytes significantly increased in the lesion area on different time courses post-TBI,and peaked on day 7.4.The level of reactive astrocytes-derived BDNF significantly increased in the lesion area on different time courses post-TBI,and peaked on day 7.The Neuroblasts(DCX-positive cells),BDNF and reactive astrocytes(GFAP-positive cells)were adjacent or co-located.5.BDNF at different concentrations(50ng/mL,100ng/mL and500ng/mL)increased the migration distance of neuroblasts in SVZ explants.BDNF at the concentration of 100ng/mL had the best effect on neuroblasts migration.Part2 Exploration of neuroblasts migration to be induced artificially.1.Artificial intervention with the appropriate concentration of TNF-α(200ng/mL),IL-6(200ng/mL)or MCP-1(50ng/mL,100ng/mL and500ng/mL)could induce the directional migration of neuroblasts,and MCP-1 could provoke reactive astrocyte to express BDNF in physiological mice.2.Artificial intervention with the appropriate concentration of TNF-α(200ng/mL)or MCP-1(50ng/mL,100ng/mL)could induce the directional migration of neuroblasts,and MCP-1 could provoke reactive astrocyte to express BDNF in mice on day 28 after TBI.3.The neuroblasts that arrived at the target area by artificial intervention with the appropriate concentration of MCP-1 might develop into mature neurons.Artificial intervention with appropriate concentration of MCP-1(50ng/mL or 100ng/mL)not only played a positive role in inducing the directional migration of neuroblasts,but also reduced the negative effect on cells in the target area.4.The level of MCP-1 significantly increased in peri-cortex at different post-CCI time points,and peaked on day 7.Conclusion1.Neuroblasts migrated from SVZ to the lesion area,and arrived at the lesion area on day 7 after TBI.The concentration gradient of reactive astrocytes-derived BDNF might be a key factor stimulating neuroblasts migration after TBI.This endogenous directional migration was significantly weak on day 21 after TBI.2.Artificial intervention with appropriate concentration of TNF-α,IL-6 or MCP-1 can induce directional migration of neuroblasts in physiological mice.Artificial intervention with appropriate concentration of TNF-α or MCP-1 can induce directional migration of neuroblasts in mice on day 28 after TBI.MCP-1 could provoke reactive astrocyte to express BDNF not only in physiological mice but also in mice on day 28 after TBI.The neuroblasts that arrived at the target area might develop into mature neurons.MCP-1 might be the endogenous regulator of neurogenesis after TBI.