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Objective To investigate the proliferative capacity of neural stem cells (NSCs) in rat hippocampus after traumatic brain injury (TBI) and its relationship with Janus kinase 2/signaling and transcriptional activation factor 3 (JAK2/STAT3) signaling pathway activity.Methods A total of 108 SD rats were randomly divided into control group (36 rats) and TBI group (72 rats).The TBI model was constructed by PinPointTM Precision Cortical Impactor.At 1,3,7,14,21 and 28 days after injury,the brain tissues were taken for immunofluorescence staining to detect the proliferation of NSCs [5-bromodeoxyuridine (BrdU) +/stem cell key protein-2 (Sox2) +] in hippocampus,and phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3 (p-STAT3) were detected by Western blot.The expression level of p-JAK2 and p-STAT3 as well as the changing trend were analyzed.On the basis of preliminary analysis of the proliferation of NSCs and the change of JAK2/STAT3 signaling pathway activity in hippocampus,another 24 SD rats were randomly divided into TBI + normal saline group and TBI +AG490 (JAK2 specific inhibitor) group,with 12 rats in each group.At 7 days after injury,the proliferation of NSCs in hippocampus was detected by immunofluorescence staining,and the expression levels of p-JAK2 and p-STAT3 were detected by Western blot,so as to further confirm the correlation between the proliferation ability of NSCs in hippocampus and JAK2/STAT3 signaling pathway.Results Compared with the control group,the number of NSCs in the hippocampus of the TBI group and the expression of p-JAK2 and p-STAT3 increased.And the most significant increase occurred at 7 days after injury [number of NSCs:31.2 ± 4.7 in the control group,111.4 ± 8.1 in the TBI group (P ＜ 0.01);p-JAK2:1.11 ± 0.09 in the control group,2.16 ± 1.01 in the TBI group (P ＜ 0.01);p-STAT3:1.05 ± 0.06 in the control group and 2.06 ± 0.09 in the TBI group (P ＜ 0.01)].The proliferation of NSCs in hippocampus of TBI group was consistent with the change of p-JAK2 and p-STAT3 expression.Seven days after injury,the expression levels of p-JAK2 and p-STAT3 and the proliferation ability of NSCs in the TBI + AG490 were significantly decreased [p-JAK2:2.18 ± 0.15 in the TBI + isotonic saline group,1.24 ±0.10 in the TBI + AG490 group (P ＜0.01);p-STAT3:2.21 ±0.12 in the TBI + isotonic saline group,1.25 ± 0.11 in the TBI + AG490 group (P ＜ 0.01);NSCs number:112.8 ± 8.6 in the TBI + isotonic saline group,75.5 ± 6.4 in the TBI + AG490 group (P ＜ 0.05)].Conclusions The proliferation of NSCs in hippocampus of rats increased after TBI,and the activity of JAK2/STAT3 signaling pathway also increased,following the same trend.JAK2 inhibitor AG490 can reduce the activity of JAK2/STAT3 signaling pathway and the proliferation of NSCs.This can provide reference for researches on TBI promoting nerve regeneration and function repair.
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Objective To explore the influence of exosome-derived miR-124 on the molecular expression related to axonal regeneration after mechanical damage to cortical neurons in mice,aiming to provide experimental data for intervention in neurogenesis after traumatic brain injury (TBI).Methods The plasmid loaded with miR-124 was used to transfect the HEK293 cell line.The transfection effect was identified by real time Polymerase Chain Reaction (qPCR).The exosomes were isolated from the supematant of cultured transfected HEK293 cell line by the SBI isolation kit.The isolated exosomes were identified by electron microscopy and Western blotting,and the involved miR-124 in the exosomes was identified by qPCR.After the cortical neurons were isolated from the pregnant mice (14-17-day old) and cultured for 7 days,they were divided into 4 groups:control,damage,damage + exosomes without miR-124 and damage + exosomes with miR-124.The Petri dishes were manually scratched with a 10 μL plastic stylet needle to construct a mechanical damage in vitro in the latter 3 groups.The isolated exosomes without or with miR-124 were added into the cultured medium for culture for 72 h in the latter 2 groups,respectively.The expression ofmiR-124,NRP-1,Tau and Gap-43 was measured by qPCR and Western blotting respectively.Results The exosomes containing miR-124 were successfully obtained by plasmid transfection and the SBI isolation kit.The expression levels of miR-124,NRP-1 and Gap-43 in the damage + exosomes with miR-124 group were elevated significantly greater than in the other 3 groups (P＜0.05).The expression levels ofmiR-124,NRP-1 and Gap-43 in the damage group and damage + exosomes without miR-124 group were elevated significantly greater than in control group (P＜0.05).Conclusions The exosomes may transmit miR-124 to the cortical neurons in mice after mechanical damage and increase the expression ofmiR-124,NRP-1 and Gap-43 in the cortical neurons in mice.
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Objective To explore the effect of exosome derived micro RNA (miR)-124 on activation status of microglia cells in injured brain tissues at acute phase of traumatic brain injury (TBI), and further provide theoretical references for intervention of neuroinflammation after TBI. Methods (1) In vitro cultured HEK293 cells were divided into miR-124 transfected group and control group, and miR-124 plasmids or Control siRNA by plasmid were transfected into the cells of the two groups;two-three weeks after isolation of monoclonal cell lines and two weeks after continuous culture, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the miR-124 content in cells of the two groups; the exosomes were extracted from the supernatant of cells from the two groups using SBI kit, and the morphology of the exosomes was observed under electron microscope; the expression of CD63, a surface marker molecule, was detected by Western blotting; RT-PCR was used to determine the miR-124 content in the exosomes of the two groups. (2) A total of 60 healthy male rats were randomly divided into sham-operated group (n=12), TBI group (n=12) and TBI+Exo-124 group (n=24); TBI models were constructed by controlled cortical injury device, and Exo-124 (3×109 particles) was given into the TBI+Exo-124 group via tail intravenous injection and equivalent solvent was given to the sham-operated group and TBI group 24 h after TBI; 3 d after modeling, RT-PCR was used to detect the miR-124 expression in brain tissues of the injured areas of the three groups; flow cytometry (FCM) was used to detect the percentages of Iba-1+/CD32+ and Iba-1+/CD206+ microglial cells in brain tissues; enzyme-linked immunosorbent assay (ELISA) was used to detect the expressions of interleukin (IL)-1, IL-6, IL-4 and IL-10 in the brain tissues. Results (1) RT-PCR showed that the miR-124 expression in the miR-124 transfected group was statistically higher than that in the control group (P<0.05); electron microscopy showed spherical particles with diameter about 100 nm and obvious membrane structure; Western blotting showed that the expression level of CD63 in the miR-124 transfected group was significantly higher than that in the control group (P<0.05); RT-PCR showed that the miR-124 content in the miR-124 transfected group was significantly higher than that in the control group (P<0.05). (2) The miR-124 expression in injured brain tissues of TBI+Exo-124 group was statistically higher than that in TBI group and sham-operated group (P<0.05); as compared with those in the sham-operated group, the percentages of Iba-1+/CD32+ and Iba-1+/CD206+ microglial cells and the expressions of IL-1, IL-6, IL-4 and IL-10 in the brain tissues of TBI group were significantly increased (P<0.05); as compared with the TBI group, the TBI+Exo-124 group had significantly decreased percentage of Iba-1+/CD32+ microglial cells and significantly increased percentage of Iba-1+/CD206+ microglial cells, statistically decreased IL-1 and IL-6 expressions, and statistically increased IL-4 and IL-10 expressions (P<0.05). Conclusion Exosome-derived miR-124 promotes the polarization of microglia cells from M1 to M2 and reduces neuroinflammation at acute phase of TBI.
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Objective To investigate the effects of sphingosine-1-phosphate receptor 1 (S1PR1) changes on the proliferation of endogenous neural stem cells (NSCs) in hippocampus after traumatic brain injury (TBI).Methods Rat TBI models were constructed by the means of controlled cortical injury.A total of 72 rats were included and randomly divided into four groups:sham,TBI,TBI + SEW (TBI + S1PR1 agonist SEW2871 intervention) and TBI + VPC group (TBI + S1PR1 antagonist VPC23019 intervention),with 18 rats per group.The TBI model was induced by a control cortical injury device.The injured rats in TBI + SEW group and TBI + VPC group were respectively administrated with S1PR1 agonist SEW2871 and antagonist VPC23019 at scheduled time points after TBI.Hippocampal S1PR1 expression was detected by Western-blotting and the proliferation of NSCs was assessed by double-labeled immunofluorescence staining at days 7,14 and 21 after injury.Results At days 7,14 and 21 after TBI,the hippocampal S1PR1 levels and NSCs proliferation amounts in sham,TBI,TBI + SEW and TBI + VPC groups were evidently different (P ＜ 0.05).In particular,the outstanding changes among the four groups above occurred at 7 d after injury were as following:S1PR1 expression in TBI group significantly increased by 1.56 times compared with that in sham group,and it was respectively upregulated by 66.67％ in TBI + SEW group and down-regulated by 20.29％ in TBI + VPC group (P ＜0.05).The nmmber of NSCs proliferation in TBI group was 2.08 times more than that in sham group,and it increased by 36.75％ in TBI + SEW group and reduced by 18.77％ in TBI + VPC group (P ＜ 0.05).Conclusion The expression of S1 PRI is closely associated with the proliferation of NSCs in hippocampus after TBI,indicating that S1PR1 activation may be an effective strategy to improve the posttraumatic neurogenesis.
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Objective To investigate the effects of sphingosine-1-phosphate receptor 1 (S1PR1) changes on the proliferation of endogenous neural stem cells (NSCs) in hippocampus after traumatic brain injury (TBI).Methods Rat TBI models were constructed by the means of controlled cortical injury.A total of 72 rats were included and randomly divided into four groups:sham,TBI,TBI + SEW (TBI + S1PR1 agonist SEW2871 intervention) and TBI + VPC group (TBI + S1PR1 antagonist VPC23019 intervention),with 18 rats per group.The TBI model was induced by a control cortical injury device.The injured rats in TBI + SEW group and TBI + VPC group were respectively administrated with S1PR1 agonist SEW2871 and antagonist VPC23019 at scheduled time points after TBI.Hippocampal S1PR1 expression was detected by Western-blotting and the proliferation of NSCs was assessed by double-labeled immunofluorescence staining at days 7,14 and 21 after injury.Results At days 7,14 and 21 after TBI,the hippocampal S1PR1 levels and NSCs proliferation amounts in sham,TBI,TBI + SEW and TBI + VPC groups were evidently different (P ＜ 0.05).In particular,the outstanding changes among the four groups above occurred at 7 d after injury were as following:S1PR1 expression in TBI group significantly increased by 1.56 times compared with that in sham group,and it was respectively upregulated by 66.67％ in TBI + SEW group and down-regulated by 20.29％ in TBI + VPC group (P ＜0.05).The nmmber of NSCs proliferation in TBI group was 2.08 times more than that in sham group,and it increased by 36.75％ in TBI + SEW group and reduced by 18.77％ in TBI + VPC group (P ＜ 0.05).Conclusion The expression of S1 PRI is closely associated with the proliferation of NSCs in hippocampus after TBI,indicating that S1PR1 activation may be an effective strategy to improve the posttraumatic neurogenesis.
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Objective To observe the expression changes of microRNA-124(miRNA-124) following traumatic brain injury (TBI) in mice and investigate the correlation of miRNA-124 with neural axon regeneration.Methods Ninety-one C57BL/6 mice were assigned into TBI group (n =63) and control group (n =28) according to the random number table.Mice in TBI group were subjected to controlled cortical impact and euthanized at 12 hours and 1,3,7,14,21,28 days postinjury for the collection of brain tissue in the trauma zone.Mice in control group underwent craniectomy only.Trauma zone observation was done using the HE staining.Expression of miRNA-124 was detected using the real-time PCR.Levels of Nrp-1,Gap-43 and Tau were detected using the Western blot and immunohistochemical staining.Results After injtury,study of mice behavior and HE staining indicated the establishment of experimental model was successful.Expression of miRNA-124 reached the peak at 3 days postinjury (3.80 ± 0.22),expression of Nrp-1 reached the peak at 7 days postinjury (2.006 ±0.179),expression of Tau reached the peak at 14 days postinjury (2.063 ±0.172),and expression of Gap-43 sustained high level since 12 hours after injury(1.355 ± 0.093) (P ＜ 0.05).Count of axon marker positive cells in TBI group was the lowest at 1 day postinjury due to the direct damage and edema,and then slowly recovered.There was no significant difference in the count of axon marker positive cells between the two groups at 14,21 and 28 days postinjury (P ＞ 0.05),but the morphology in TBI group changed obviously.Although the positive cells of axon marker decreased at 1 day postinjury,expressions of miRNA-124,Nrp-1,Tau and Gap-43 in TBI group were significantly increased compared to the detections in control group (P ＜ 0.05).Conclusion Increased expression of miRNA-124 in trauma zone may closely related to axon regeneration after TBI in mice.