Ectoderm-derived frontal bone mesenchymal stem cells promote traumatic brain injury recovery by alleviating neuroinflammation and glutamate excitotoxicity partially via FGF1

Abstract

Background: Traumatic brain injury (TBI) leads to cell and tissue impairment, as well as functional deficits. Stem cells promote structural and functional recovery and thus are considered as a promising therapy for various nerve injuries. Here, we aimed to investigate the role of ectoderm-derived frontal bone mesenchymal stem cells (FbMSCs) in promoting cerebral repair and functional recovery in a murine TBI model.

Methods: A murine TBI model was established by injuring C57BL/6 N mice with moderate-controlled cortical impact to evaluate the extent of brain damage and behavioral deficits. Ectoderm-derived FbMSCs were isolated from the frontal bone and their characteristics were assessed using multiple differentiation assays, flow cytometry and microarray analysis. Brain repairment and functional recovery were analyzed at different days post-injury with or without FbMSC application. Behavioral tests were performed to assess learning and memory improvements. RNA sequencing analysis, immunofluorescence staining, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to examine inflammation reaction and neural regeneration. In vitro co-culture analysis and quantification of glutamate transportation were carried out to explore the possible mechanism of neurogenesis and functional recovery promoted by FbMSCs.

Results: Ectoderm-derived FbMSCs showed fibroblast like morphology and osteogenic differentiation capacity. FbMSCs were CD105, CD29 positive and CD45, CD31 negative. Different from mesoderm-derived MSCs, FbMSCs expressed the ectoderm-specific transcription factor Tfap2β. TBI mice showed impaired learning and memory deficits. Microglia and astrocyte activation, as well as neural damage, were significantly increased post-injury. FbMSC application ameliorated the behavioral deficits of TBI mice and promoted neural regeneration. RNA sequencing analysis showed that signal pathways related to inflammation decreased, whereas those related to neural activation increased. Immunofluorescence staining and qRT-PCR data revealed that microglial activation and astrocyte polarization to the A1 phenotype were suppressed by FbMSC application. In addition, FGF1 secreted from FbMSCs enhanced glutamate transportation by astrocytes and alleviated the cytotoxic effect of excessive glutamate on neurons.

Conclusions: Ectoderm-derived FbMSC application significantly alleviated neuroinflammation, brain injury, and excitatory toxicity to neurons, improved cognition and behavioral deficits in TBI mice. Therefore, ectoderm-derived FbMSCs could be ideal therapeutic candidates for TBI which mostly affect cells from the same embryonic origins as FbMSCs.

Keywords: Frontal bone mesenchymal stem cells; Glutamate excitotoxicity; Neuroinflammation; Traumatic brain injury.

Fig. 1

Characteristics of frontal bone mesenchymal stem cells (FbMSCs). a Schematic of murine skull. Areas marked F refer to frontal bone, from where FbMSCs were isolated. b Representative morphological features of FbMSCs. Scale bar, 200 μm. c ALP staining of FbMSCs. Scale bar, 50 μm. d Flow cytometric analysis of FbMSCs. e Expression of iNOS, IL-10, IL6, and HGF in FbMSCs stimulated with the indicated factors. Microarray analysis was performed on FbMSCs and bone marrow MSCs (BMSCs). f Cluster heat map of representative differential genes in FbMSCs and BMSCs. GO (g) and KEGG (h) analysis of enriched pathway in FbMSCs. Quantitative RT-PCR verified the higher expression of Tfap2β, Fgf1, and Grm1 (i), as well as genes related to the neuron ligand-receptor pathway (j), axon guidance pathway and MAPK pathway (k) in FbMSCs. (Data are presented as the mean ± standard error; *, **, ***, and **** indicate significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively).

Fig. 2

FbMSC transplantation improved learning and cognitive ability in TBI mice. a Experimental schedule. FbMSCs were transplanted immediately after TBI (D0). All mice underwent behavioral tests at the indicated days. b Schematic of beam walk test (left) and latency to cross the beam (right). c–f Morris water maze test was used to evaluate the learning and cognitive ability of mice. Typical escape route map (c), learning curve (d), escape latency (e), and movement speed (f) of each group were displayed. Total distance (g) and mean speed (h) of each group in the open field test. i Schematic (left) and results (right) of novel object recognition experiment. (n = 8–10 mice per group; Data are presented as the mean ± standard error; *, **, ***, and **** indicate significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively).

Fig. 3

RNA sequencing analysis of FbMSC-treated TBI group and TBI group. Representative photos of wound healing (a) and topical views of brains (b) from the indicated groups collected at days 4, 14, and 28 after TBI. Scale bar, 2 mm. Volcano maps (c) and heat map (d) of differentially expressed genes in FbMSC-treated TBI group and TBI group. e GO analysis showed enrichment in cellular component, molecular function and biological process. f KEGG analysis of enriched pathway in FbMSCs. Quantitative RT-PCR verified the expression genes related to neuroactive ligand-receptor interaction (g), IL-17 signaling pathway (h) and MAPK signaling pathway (i). (n = 3–4 mice per group; Data are presented as the mean ± standard error; *, **, ***, and **** indicate significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively).

Fig. 4

FbMSC application promotes the recovery of axonal injury in TBI mice. MAP2 immunofluorescence and fluorescence intensity quantification in peri-impact area and hippocampus in each group at 4 d (a, b) and 28 d (c, d) post-injury. Scale bar, 20 μm. e Representative 3D reconstruction images of MAP2 staining in hippocampus in each group at 4 d post-injury. Scale bar, 150 μm. Doublecortin (DCX) immunofluorescence image (f) and fluorescence intensity quantification (g) in each group at 14 d post-injury. Scale bar, 20 μm. h Expression of BDNF was detected by qRT-PCR at 14 d post-injury. (n = 3–4 mice per group; Data are presented as the mean ± standard error; *, **, ***, and **** indicate significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.)

Fig. 5

FbMSC application decreased the activation of microglia in TBI mice. a Representative Iba1-stained coronal sections of murine brains of the four groups at 4 d post-injury. Scale bar, 2 mm. Expression of Aif1, Cx3cr1 (b) and pro-inflammatory factors TNFα, IL1β, IL6 (c) was detected by qRT-PCR at 4 d, 14 d, and 28 d post-injury. Iba1 immunofluorescence and mean fluorescence intensity quantification in peri-impact area and hippocampus in each group at 4 d (d, e) and 28 d (f, g) post-injury. Scale bar, 20 μm. (n = 3–4 mice per group; Data are presented as the mean ± standard error; *, **, ***, and **** indicate significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.)

Fig. 6

Application of FbMSCs decreased the activation of astrocytes. a Representative GFAP stained coronal sections of murine brains of the four groups at 4 d post-injury. Scale bar, 2 mm. b Expression of H2D1 and GBP2 was detected by qRT-PCR at 4 d, 14 d, and 28 d post-injury. GFAP immunofluorescence and fluorescence intensity quantification in peri-impact area and hippocampus in each group at 4 d (c, d) and 28 d (e, f) post-injury. Scale bar, 20 μm. g Schematic of FbMSCs and astrocyte cell line C8-D1A co-culture. h C8-D1A cells were collected after 24 h co-culture. IL6, IL1β, and TNFα expression was detected by qRT-PCR. i IL6, IL1β and TNFα expression was detected after TNFα (150 ng/mL), IL1β (50 ng/mL) and FGF1 (1, 10, 100 ng/mL) induction. (n = 3–4 mice per group; Data are presented as the mean ± standard error; *, **, ***, and **** indicate significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively).

Fig. 7

FGF1 from FbMSCs promotes glutamate uptake of C8-D1A cells and alleviates neuron excitotoxicity. a Representative images of MAP2 stained neurons treated with indicated doses of glutamate. Scale bar, 20 μm. Branch point (b) and Dendritic length (c) were quantified according to MAP2 staining. Representative staining images (d) and quantification of cell viability (e) according to calcein-AM (green)/ethidium homodimer (red) staining on HT22 cells. Scale bar, 100 μm. f Glutamate uptake of C8-D1A cells stimulated with indicated doses of FGF1. g Glutamate uptake of C8-D1A cells stimulated with indicated dose of TNFα (150 ng/mL), IL1β (50 ng/mL) and FGF1 (50 ng/mL). Representative images (h) and quantification of neuron branches (i) of MAP2-stained neurons in indicated groups. Scale bar, 20 μm. Representative images (j) and quantification of cell viability (k) of calcein-AM (green)/ethidium homodimer (red) staining of HT22 cells in the indicated groups. Scale bar, 100 μm. l Expression level of FGF1 mRNA in FbMSCs after transfected with siFGF1 and siNC. m Western blot analysis demonstrated levels of FGF1 after transfected with siFGF1 and siNC. n Western blot analysis demonstrated levels of BDNF in lesion areas of mice at 4 days. o mRNA level of BDNF and NGF was detected by qRT-PCR. p Representative images of active caspase-3 staining in the lesion areas from each group at 4 days. Scale bar, 20 μm. (n = 3–4 mice per group; Data are presented as the mean ± standard error; *, **, ***, and **** indicate significance at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.)

Fig. 8

Proposed mechanism. FbMSC application reduces the activation of microglia cells and astrocytes and alleviates glutamate excitotoxicity. Abbreviations: FGF1, acidic fibroblast growth factor1; FGFR, acidic fibroblast growth factor1 receptor; GLT-1, glutamate transporter-1; GLAST, glutamate-aspartate transporter; iNOS, inducible nitric oxide synthase; IL6, interleukin 6; IL-10, interleukin-10