Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats.

It was not clear how and whether neural stem cells (NSCs) responded to toll-like receptor 2 (TLR2) in the inflammatory environment after traumatic brain injury (TBI). The current study investigated the correlation of TLR2 and NSC proliferation in the dentate gyrus (DG) using the TBI model of rats. Immunofluorescence (IF) was used to observe the expression of BrdU, nestin, and TLR2 in the DG in morphology. Proliferating cells in the DG were labelled by thymidine analog 5-bromo-2-deoxyuridine (BrdU). Three-labelled BrdU, nestin, and DAPI was used for the identification of newly generated NSCs. Western blotting and real-time polymerase chain reaction (PCR) were used to observe the expression of TLR2 from the level of protein and mRNA. We observed that BrdU+/nestin+/DAPI+ cells accounted for 84.30% ± 6.54% among BrdU+ cells; BrdU+ and nestin+ cells in the DG were also TLR2+ cells. BrdU+ cells and the expression of TLR2 (both protein and mRNA levels) both elevated immediately at 6 hours (h), 24 h, 3 days (d), and 7 d posttrauma and peaked in 3 d. Results indicated that TLR2 was expressed on proliferating cells in the DG (NSCs possibly) and there was a potential correlation between increased TLR2 and proliferated NSCs after TBI. Taken together, these findings suggested that TLR2 was involved in endogenous neurogenesis in the DG after TBI.

MiR-124 Enriched Exosomes Promoted the M2 Polarization of Microglia and Enhanced Hippocampus Neurogenesis After Traumatic Brain Injury by Inhibiting TLR4 Pathway.

MicroRNA-124 (miR-124) is a brain specific miRNA that is highly expressed in microglia. The upregulation of miR-124 contributes to M2 polarization of microglia, which is beneficial to neurogenesis. Exosomes are lipid membrane vesicles that can deliver miR-124 into the brain. However, whether miR-124 enriched exosomes (Exo-miR-124) can regulate the polarization of microglia and affect hippocampus neurogenesis after traumatic brain injury (TBI) is unknown. To clarify this, the Exo-miR-124 was first constructed, and then was intravenously administrated into rats via tail vein with the dose of 3 × 109 particles/each rat at 24 h post TBI. The polarization of microglia in hippocampus was evaluated through measuring the signature genes and cytokines of M1/M2 phenotype by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immune sorbent assay (ELISA) at 7/14/21/28 days after TBI. Hippocampus neurogenesis was evaluated through detecting the proliferation marker BrdU/SOX2 and differentiation marker BrdU/NeuN by immunofluorescence (IF) at 7 and 28 days after TBI respectively. Neurological function was evaluated by neurological severity score (NSS) and morris water maze (MWM) at 7/14/21/28 and 24-28 days after TBI respectively. To explore the underlying mechanisms, the mRNA expression of TLR4 pathway molecules in hippocampus were measured by RT-PCR, and the polarization of microglia and the activation of TLR4 pathway in BV2 cells were measured after exosome treatment as well. Results demonstrated that Exo-miR-124 treatment promoted the M2 polarization of microglia, enhanced neurogenesis in hippocampus, and improved function recovery after TBI. The M2 polarization effect of Exo-miR-124 was produced through inhibiting TLR4 pathway, which was verified in hippocampus and BV2 microglia. In conclusion, Exo-miR-124 treatment promoted M2 polarization of microglia and improved hippocampal neurogenesis and functional recovery after brain injury, which might be a strategy to improve the outcome of TBI.

The Effect of SPTLC2 on Promoting Neuronal Apoptosis is Alleviated by MiR-124-3p Through TLR4 Signalling Pathway.

To investigate the role and mechanism of microRNA-124-3p (miR-124-3p) and serine palmitoyltransferase long chain base subunit 2 (SPTLC2) in neuronal apoptosis induced by mechanical injury. Transient transfection was used to modify the expression of miR-124-3p and SPTLC2. After transfection, neuronal apoptosis was evaluated in an in vitro injury model of primary neurons using TUNEL staining and western blot. The correlation between miR-124-3p and SPTLC2 was identified through a dual luciferase reporter assay in HEK293 cells. A rescue experiment in primary neurons was performed to further confirm the result. To explore the downstream mechanisms, co-immunoprecipitation was performed to identify proteins that interact with SPTLC2 in toll-like receptor 4 (TLR4) signalling pathway. Subsequently, the relative expression levels of TLR4 pathway molecules were measured by western blot. Our results showed that increased miR-124-3p can inhibit neuronal apoptosis, which is opposite to the effect of SPTLC2. In addition, miR-124-3p was proved to negatively regulate SPTLC2 expression and suppress the apoptosis-promoting effect of SPTLC2 via the TLR4 signalling pathway.

Acute Traumatic Brain Injury Induces CD4+ and CD8+ T Cell Functional Impairment by Upregulating the Expression of PD-1 via the Activated Sympathetic Nervous System.

OBJECTIVE: Traumatic brain injury (TBI) induces immunosuppression in the acute phase, and the activation of the sympathetic nervous system (SNS) might play a role in this process, but the mechanism involved is unknown. Herein, we explored the impact of acute (a)TBI on the peripheral immune system and its correlation with the SNS and the T cell exhaustion marker, PD-1 (programmed cell death-1). METHODS: Flow cytometry (FCM) was performed to analyze the expression of T cell markers and intracellular cytokines, interferon-γ and tumor necrosis factor-α, and the T cell exhaustion marker, PD-1, in the peripheral blood mononuclear cells (PBMCs) of TBI rats. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze the concentration of norepinephrine (NE) in the serum. Propranolol was administrated to block the SNS in vivo and NE stimulation was used to imitate the activation of the SNS in vitro. RESULTS: We found that the concentration of NE was significantly elevated after TBI, and the dysfunction of CD4+ and CD8+ T cells was reversed by the SNS blocker propranolol in vivo and imitated by the SNS neurotransmitter NE in vitro. The expression of PD-1 on CD4+ and CD8+ T cells was upregulated after aTBI, which was reversed by propranolol administration in vivo and imitated by NE stimulation in vitro. Furthermore, the PD-1 blocker reversed the dysfunction of CD4+ and CD8+T cells in vitro. CONCLUSION: Our findings demonstrated that aTBI activated the SNS, and further upregulated the expression of PD-1 on CD4+ and CD8+ T cells, which, in turn, impaired their function and contributed to immunosuppression.

[Activation of miR-124-3p/Notch pathway promotes proliferation and differentiation of rat neural stem cells after traumatic brain injury].

Objective To explore the change of the expression of microRNA-124-3p (miR-124-3p) in injured hippocampus of rats and investigate the role of miR-124-3p in neuranagenesis after traumatic brain injury (TBI). Methods The healthy male rats were randomly divided into a sham-operated group, TBI group, miR-124-3p agomir group and miR-124-3p antagomir group. TBI models were constructed by controlled cortical injury (CCI) device for all the groups except for the sham-operated group. The miR-124-3p agomir (1 nmol) was given to the miR-124-3p agomir group and miR-124-3p antagomir (1 nmol) to the miR-124-3p antagomir group via lateral ventricular injection, and equivalent solvent was given to the sham-operated group and TBI group after injury. The injured hippocampus of rats was collected at 12 hours, 1 day, 3, 7 days after injury. The real-time PCR and Western blot analysis were used to examine the expression of miR-124-3p and Delta-like 1 (DLL1) in the injured hippocampus. Immunofluorescence histochemistry was used to examine the expression levels of 5-bromodeoxyuridine (BrdU), neuronal nuclear antigen (NeuN) and nestin in the injured hippocampus. Bioinformatics software was used to predict and dual luciferase reporter assay to validate the regulatory relationship between miR-124-3p and DLL1. Results The miR-124-3p and DLL1 expression in the TBI group were significantly higher than those in the sham-operated group; compared with the TBI group, the miR-124-3p agomir group had significantly increased expression of miR-124-3p and significantly decreased expression of DLL1 in the injured hippocampus, and miR-124-3p antagomir group had significantly decreased expression of miR-124-3p and significantly increased expression of DLL1. Compared with the sham-operated group, the BrdU+NeuN+ cells and BrdU+nestin+ cells in the hippocampus significantly increased in the TBI group at 7 days after injury. The miR-124-3p agomir treatment increased the number of the BrdU+NeuN+ cells and BrdU+nestin+ cells, while the miR-124-3p antagomir treatment decreased the number of the BrdU+NeuN+ cells and BrdU+nestin+ cells. Bioinformatics analysis confirmed that DLL1 was a target of miR-124-3p. Conclusion High expression of miR-124-3p in the trauma region promotes the proliferation and differentiation of neural stem cells by targeting and inhibiting DLL1.