It has previously been demonstrated that bone marrow stromal cells (BMSCs) exhibit great therapeutic potential in neuronal injuries; however, there is limited understanding of the precise underlying mechanisms that contribute to functional improvement following brain injury. The aim of the present study was to assess the effect of BMSC treatment on traumatic brain injury (TBI) in rats, and investigate if they migrate to injured areas and promote neuromotor functional recovery via upregulation of neurotrophic factors and synaptic proteins. BMSCs were cultured
Traumatic brain injury (TBI) is a primary health concern and leading cause of mortality worldwide. Neurological deficits resulting from TBI present a severe burden to patient families and society. There is no current effective treatment method to promote functional recovery except for routine rehabilitation, hyperbaric oxygen and basic care. It has previously been demonstrated that neural stem cells (NSCs) may promote neurological recovery following brain injury (
Previous studies have revealed a significant loss of synapses in the days following brain injury, notably in the brain regions connected to the site of initial injury, including the hippocampus (
The present study hypothesized that the effect of BMSCs on motor function may be associated with the expression of synaptophysin (SYN). Therefore, a rat model of TBI was constructed to investigate if BMSCs migrate to injured areas and promote functional recovery via upregulation of neurotrophic factors and synaptic proteins.
A total of 15 Sprague-Dawley (SD) female rats (age, 1 month; weight, 20–24 g), were obtained from the Hebei Medical University Experimental Animal Center (Shi Jiazhuang, China) and were housed in a temperature-controlled (22–24°C) room with a 12-h light/dark cycle and with water and food freely available. Rats were anaesthetized with 10% chloral hydrate (3 ml/kg; Bio-Rad Biotechnology, Inc., Shanghai, China) and BMSCs were isolated and cultured. Briefly, fresh bone marrow cells were collected from the femurs of SD rats, via suction from the medullary cavity using a 20-ml sterile syringe. All rats were sacrificed following cell harvesting. For anticoagulation, 5 ml heparin (100 IU/ml) was used. Following filtration, cells were centrifuged at 1,000 × g for 5 min at 4°C. The purified cells were cultured in a 25 cm2 flask with Dulbecco's modified Eagle's medium/nutrient mixture F12 (DMEM/F12; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare, Logan, UT, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), and incubated at 37°C in 5% CO2. After 48 h, non-adherent cells were removed and fresh media was added. When adherent cells reached ~80% confluency, the cells were dissociated with 0.25% trypsin solution and re-seeded again. Following three passages of culture, passage 3 BMSCs were used for subsequent experiments.
Adult male SD rats (weight, 250–300 g; age, 12–16 weeks), were obtained from Hebei Medical University Experimental Animal Center and were housed in a temperature-controlled (22–24°C) room with a 12-h light/dark cycle and with water and food freely available. A total of 105 rats were utilized in this study. All experimental procedures were performed in accordance with the guidelines of the Chinese council on animal protection, and were approved by the Hebei Medical University Committee (Shijiazhuang, China) for the use of animals in research (Permit Number: 2015046). The TBI model was developed as described using a weight-drop device (
The 105 adult rats were randomly divided into 3 groups (n=35/group): Sham, TBI and TBI + BMSC-treated. Prior to transplantation, BMSCs were digested with trypsin, washed twice with DMEM and centrifuged at 1,000 × g for 5 min at 4°C. In BMSC-transplanted animals, the BMSCs (3×106 cells/ml) in 1 ml of phosphate-buffered saline (PBS) were transplanted via a tail vein puncture into rats 30 min following the induction of TBI. The sham and TBI groups received equal volumes of saline injection. Each subgroup was composed of five rats, and rats were anesthetized with 10% chloral hydrate and decapitated 1, 3, 5, 7 and 14 days following TBI. The remainder of the rats (n=10 per treatment group) underwent neurobehavioral examinations. All investigations were blind and the groups were revealed at the end of the behavioral and histological analyses.
A rotarod was used to assess motor function as previously described (
Neurological deficits were evaluated using the mNSS on an 18-point scale by a researcher blinded to treatment, which tested reflexes, alertness, coordination and motor abilities. One point was awarded for failure to perform a particular task; thus, the higher the score, the more severe the injury, whereas a healthy rat scored zero. Post-injury, mNSS was evaluated at days 1–14 post-TBI.
Brain tissues were fixed in 4% paraformaldehyde solution for 24 h, washed with running water for 4 h, and embedded in paraffin and dehydrated with gradient alcohol and xylene. The samples were serially sectioned at a thickness of 5 µm. All sections were mounted on glass slides and subsequently stained with hematoxylin and eosin (H&E). Sections were observed and analyzed using an optical microscope.
Rats were decapitated under deep anesthesia and the brains were rapidly isolated. The hippocampal tissues were dissected on ice, the proteins were extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) from the cortex surrounding the injured area and the protein concentration was determined using a bicinchoninic acid kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Samples (50 µg) were separated by 10% SDS-PAGE and subsequently transferred onto polyvinylidene membranes (Roche Diagnostics GmbH, Mannheim, Germany). The blots were blocked with 5% fat-free dry milk for 2 h at room temperature, followed by incubation with the following rabbit primary antibodies: Anti-vascular endothelial growth factor (VEGF) polyclonal (1:1,000; Abcam, ab53465, Cambridge, UK), anti-brain derived neurotrophic factor (BDNF) monoclonal (1:1,000; Abcam, ab216443) and anti-β-actin monoclonal (1:1,000; Affinity Biologicals Inc., AF7018, Ancaster, ON, Canada) at 4°C overnight. Following this, the membranes were incubated with horse-radish peroxidase (HRP)-conjugated donkey anti-mouse immunoglobulin (Ig)-G or donkey anti-rabbit IgG (sc-2314 and sc-2313; 1:5,000; both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) secondary antibodies at 37°C for 1 h. Signals were detected by Enhanced Chemiluminescence using a Western Lightning® Plus-ECL kit (Perkin-Elmer, Inc., Waltham, MA, USA). (Densitometric analysis for the blots was performed using National Institutes of Health Image software version 1.41 (Bethesda, MD, USA).
Rats were perfused transcardially with saline under deep anesthesia, followed by 4% paraformaldehyde for 24 h, and placed into 30% sucrose solution (0.1 M PBS, pH 7.4) until they sank to the bottom. The brain tissues were embedded in optimum cutting temperature compound and cut into 15 µm-thick sections coronally from the anterior to posterior hippocampus (bregma −2.0 to −3.0 mm) using a cryostat. Frozen sections were sliced with a microtome, treated with 0.4% Triton X-100 for 20 min and blocked at room temperature in normal donkey serum (017–000-121; Shanghai Solarbio Bioscience & Technology Co., Ltd., Shanghai, China) for 2 h. For immunohistochemical analyses, sections were incubated overnight at 4°C with rabbit anti-VEGF (1:100) and rabbit anti-BDNF polyclonal antibodies (1:100), and subsequently with HRP-conjugated anti-rabbit IgG antibodies at 37°C in the dark for 30 min. 3,3′-Diaminobenzidine was used to reveal the immunohistochemical reaction. For double labeling, the frozen sections were incubated with rabbit anti-sex determining region Y (SRY) polyclonal (1:100; Abcam, ab209858), mouse anti-neuronal nuclear antigen (NeuN) monoclonal (1:100; EMD Millipore, Billerica, MA, USA, MAB324-K) or anti-glial fibrillary acidic protein (GFAP) monoclonal (1:100; EMD Millipore, IF03L) antibodies overnight at 4°C. The following day, the sections were incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies (sc-2090 and sc-2099; 1:1,000; Santa Cruz Biotechnology, Inc.) for 2 h at 37°C in the dark. All cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). PBS was substituted for the primary antibody as the negative control. Sections were imaged under a laser scanning confocal microscope (Olympus Fluoview™ FV1000; Olympus Corporation, Tokyo, Japan).
All experiments were repeated three times and similar results were obtained. Statistical analysis was performed using SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation and the significance of the experimental results was determined using one-way analysis of variance followed by the Student-Newman-Keuls post hoc multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.
To determine the neuroprotective effects of BMSCs against TBI-induced brain damage, the present study examined the effects of BMSCs on motor deficits via a rotarod task and mNSS score following TBI at 1, 3, 5, 7 and 14 days. As presented in
H&E staining was performed to examine the effect of BMSCs on ipsilateral cerebral cortex neuronal damage 7 days following TBI. In the TBI rats, there were marked morphological alterations in the cortex compared with the sham group. Neuronal cell body swelling and disorder was observed, in addition to intercellular broadening, cell loss and nuclear pyknosis and karyolysis in the TBI group (
The expression of VEGF and BDNF was detected at 14 days following TBI via immunochemistry and western blot assay. As presented in
BMSCs were tracked to evaluate their migration and distribution patterns in rats. BMSCs isolated from female rats were injected into male rats
To further investigate the BMSC underlying mechanisms of action, western blotting was performed to examine the expression of SYN at 1, 3, 5, 7 and 14 days post-TBI (
The results from the present study are similar to the findings of previous investigations, indicating that in animal models of TBI or stroke, BMSCs may effectively reduce brain damage and improve functional recovery (
TBI is a highly complex disorder, resulting from injury to primary and secondary brain signaling pathways. Currently, there is no effective treatment for brain injury to promote functional recovery, except for routine rehabilitation and basic care. Notably, BMSCs have previously been demonstrated to improve neurological functional recovery in experimental TBI models. There are various explanations regarding the broad underlying mechanisms by which BMSCs exert their beneficial effects. Previous findings indicated that injected BMSCs cross the blood brain barrier and actively migrate to sites of tissue damage (
An increase in the expression level of neurotrophic factors is considered as one of the primary underlying mechanisms to promote neuroprotection and neurorepair following damage (
In addition, the present study demonstrated that BMSC treatment significantly increased the expression of synaptophysin at 1, 3 and 5 days compared with the TBI group. Synaptophysin has been extensively used as a marker protein to quantify the number of synapses during neuroanatomical remodeling, or following injury (
bone marrow stromal cells
modified neurologic severity score
vascular endothelial growth factor
brain derived neurotrophic factor
sex determining region Y
synaptophysin
4′,6-diamidino-2-phenylindole
BMSC treatment improves motor deficits. Motor dysfunction was assessed at 1, 3, 5, 7 and 14 days following induction of TBI. The TBI group rat had shorter times than the sham group rats on days 1, 3, 5 and 7 post-trauma. TBI rats regained the function of the sham animals by day 14. The BMSC group rats had longer times than TBI rats on days 1, 3, and 5 post-trauma. Data are expressed as the mean ± standard deviation. *P<0.01 vs. sham group; #P<0.05 vs. TBI group. TBI, traumatic brain injury; BMSCs, bone marrow stromal cells.
Effect of BMSCs on mNSS score. The mNSS of rats in the TBI group were significantly increased in comparison with the sham group at 1, 3, 5 and 7 days, and administration of BMSCs significantly improved neuromotor function at 1, 3 and 5 days following TBI, as reflected by a decrease in mNSS. Data are expressed as the mean ± standard deviation. *P<0.01 vs. sham group; #P<0.05 vs. TBI group. TBI, traumatic brain injury; BMSCs, bone marrow stromal cells; mNSS, modified neurological severity score.
Effect of BMSCs on cortex neuronal damage assessed via hematoxylin and eosin staining. (A) Representative staining in ipsilateral cerebral cortex areas of sham, TBI and BMSCs treatment groups at 7 days following TBI. Scale bar, 100 and 20 µm. (B) Quantification of the number of viable neurons/mm hippocampal area in each group. Data are expressed as the mean ± standard deviation (n=5/per group). *P<0.01 vs. sham group; #P<0.05 vs. TBI group. TBI, traumatic brain injury; BMSCs, bone marrow stromal cells.
Effect of BMSCs on the expression of VEGF and BDNF in the ipsilateral cerebral cortex. (A) The expression of VEGF and BDNF was determined via immunohistochemical staining. Scale bar, 50 µm. (B) Western blot images representing protein expression levels of VEGF and BDNF in the ipsilateral cerebral cortex of rats at 14 days. (C) Densitometry analysis of VEGF and BDNF bands corresponding to β-actin. *P<0.05 vs. TBI group. TBI, traumatic brain injury; BMSCs, bone marrow stromal cells; VEGF, vascular endothelial growth factor; BDNF, brain derived neurotrophic factor.
BMSCs may migrate to injured areas and differentiate into neurons or astrocytes. (A) Representative images of co-localization of SRY and NeuN or GFAP at 14 days following TBI, as determined via immunofluorescent staining and counterstaining cell nuclei with DAPI. Orange labeling in images indicates co-localization. Scale bar=50 µm. (B) Quantification of results demonstrating that the BMSC-treated group exhibited significantly greater NeuN or GFAP-positive cells co-labeled with SRY in the ipsilateral cortex compared with the TBI group. Data are expressed as the mean per field of view (n=5/group). *P<0.01 vs. TBI group. TBI, traumatic brain injury; BMSCs, bone marrow stromal cells; SRY, sex determining region Y; NeuN, neuronal nuclear antigen; GFAP, glial fibrillary acidic protein.
Effect of BMSCs on synapse protein expression. (A) Western blot images of protein expression levels of SYN in the ipsilateral cortex at 1–14 days following TBI or sham surgery. (B) Densitometry analysis of SYN bands corresponding to β-actin. Data are expressed as the mean ± standard deviation. *P<0.01 vs. sham group; #P<0.05 vs. TBI group. TBI, traumatic brain injury; BMSCs, bone marrow stromal cells; SYN, synatophysin.