DOI: https://doi.org/10.21203/rs.3.rs-880133/v1
Background: Spinal cord injury (SCI) typically results in a devastating loss of neurological function below the level of injury. Although many strategies show considerable potential for SCI treatment, the therapeutic efficacy is limited. Here, we used a mouse model of thoracic contusive SCI to investigate whether the combination of bone marrow mesenchymal stem cells (BMMSCs) transplantation and exercise training has a synergistic effect on functional restoration.
Methods: BMMSCs were injected directly into the contusion epicenter immediately after SCI, and the mice started treadmill training (TMT) 3 days after SCI. Locomotor function was evaluated by the Basso Mouse Scale (BMS), horizontal ladder test, and footprint analysis. Histological examination, transmission electron microscopy observation, immunofluorescence staining and western blotting were performed 8 weeks after SCI to further explore the potential mechanism of the synergistic repair effect.
Results: The combination of BMMSCs transplantation and TMT showed the best therapeutic effect on motor function recovery compared with the other treatment groups. Further investigations revealed that the combination of BMMSCs transplantation and TMT markedly reduced fibrotic scar tissue, protected neurons, promoted axon and myelin regeneration, and increased synapse formation to a larger extent than either TMT or BMMSCs transplantation alone. Additionally, the synergistic effects of BMMSCs transplantation and TMT on SCI recovery occurred via activation of the PI3K/AKT/mTOR pathway.
Conclusions: These findings suggest that BMMSCs transplantation combined with exercise training represents a promising combinatorial strategy to facilitate clinically meaningful recovery after SCI.
Spinal cord injury (SCI) is a serious and complex disease that can cause sensory and motor loss below the neurological level of injury [1, 2]. According to quality country-level incidence studies of SCI, approximately 250,000 to 500,000 new cases occur each year [3, 4]. Severe SCI typically leads to lifelong disability and enormous health care burdens [5]. The pathophysiology of spinal cord injury can be divided into primary and secondary injuries [6].The primary injury is the initial trauma to neurons, glial cells and their surrounding vasculature caused by initial mechanical forces such as compression, shearing, and stretching [7]. After the primary injury event, a cascade of secondary events, including apoptosis of neurons, glial scarring, demyelination, excitotoxicity, and the release of proinflammatory factors, can further exacerbate neurological dysfunction and expand the zone of tissue injury [8].
To date, many neuroregenerative strategies have been investigated as potential therapies for SCI. Previous studies have demonstrated that transplantation of bone marrow mesenchymal stem cells (BMMSCs) shows considerable potential for promoting tissue repair and functional improvement following SCI [9–12]. The most common mechanisms include neuroprotection, immunomodulation, neuronal relay formation, axonal regeneration, and remyelination [13, 14]. Although BMMSCs show therapeutic promise according to preclinical findings, clinical trials still fail to demonstrate functional recovery and neural circuit restoration [15]. A randomized study tested the clinical efficacy of autologous BMMSCs transplantation in subacute SCI patients, and the results showed that BMMSCs transplantation into the injury site of SCI patients can be performed safely, but effects on motor function have not yet been observed [16]. Many other clinical trials have also failed to report satisfactory clinical improvements [17–19]. Stem cell grafts alone showed weak efficacy in functional recovery in human SCI. One possibility is that the transplanted cells could not form a sufficient number of functional connections and failed to sustain stable neuronal relays with the host circuitry for a long time [20]. Rehabilitation training seems to be an effective approach for locomotor function recovery after SCI, which may be related to the plasticity of spinal neurons below the level of injury, corticospinal tract growth and enhancement of neurotrophic factor production [21–28]. Studies in clinical trials have also shown that functional recovery after exercise is related to the degree of activation of the motor cortex [29–31]. The permanent loss of function after SCI is caused by interruption of the ascending sensation and descending input of the spinal cord as well as failure of axon regeneration and neural circuit reconstruction [32]. A large body of evidence shows that the formation and remodeling of functional neural circuits depends on strengthening neuroplasticity [33, 34]. In summary, BMMSC grafts or rehabilitation alone may target a single aspect of SCI. Therefore, the combination of cell transplantation and exercise training may be more effective than individual therapy in SCI treatment.
The purpose of this study was to determine whether the combination of BMMSCs transplantation together with exercise training can improve the therapeutic efficacy of a single treatment in mice after thoracic contusive SCI, and to investigate the potential mechanisms of combinatorial therapies in SCI repair.
Four-week-old mice were sacrificed by cervical dislocation, and the femur and tibia were obtained with sterile forceps and surgical scissors. After the ends of the long bones were cut away, the bone marrow cavity was rinsed with minimal essential medium (alpha-MEM, Gibco, CA, USA) using a 1-ml syringe. The obtained cells were inoculated into tissue culture flasks and cultured in a humidified incubator at 37°C with 5% CO2. Nonadherent cells were removed by changing the media, and the remaining adherent cells were passaged at 70–80% confluence. BMMSCs of passages 3–5 were used in this study. Flow cytometry analysis (LSRFortessa, BD Bioscience) was used for BMMSCs identification. Passage 3 cells were stained with the following fluorescent-conjugated antibodies: PE-CD29, FITC-CD34, APC-CD44, and PerCP/CY5.5-CD45.
Sixty-five 8-week-old female C57BL/6 mice were purchased from Chengdu Dossy Inc (China). The mice were housed under specific pathogen-free (SPF) conditions in the West China Experimental Animal Center at Sichuan University. The animal study was approved by the Laboratory Animal Ethics Committee of the West China Hospital of Sichuan University, and all animal procedures were conducted according to Chinese national guidelines for the care and use of laboratory animals (Ethical approval number: 20211198A).
The mice were first anesthetized with 3% isoflurane for induction, and then anesthesia was maintained under 1.5% isoflurane during all surgical procedures. After laminectomy at T10, the mice were subjected to contusive SCI at the same level using an Infinite Horizon impactor (70 kilodyne, Precision Systems & Instrumentation). After suturing the surgical incisions, penicillin (50,000 U/kg/day) was administered intramuscularly for 3 days, and manual bladder expression was performed twice a day until spontaneous voiding returned. The sham group (n = 12) underwent only laminectomy, and the spinal cord remained intact. Forty-eight surviving SCI mice were randomized to the control, TMT, BMMSCs, and combination therapy (BMMSCs + TMT) groups (n = 12 each).
After SCI, 5 µl of phosphate-buffered saline (PBS) or BMMSCs (1 × \({10}^{5}\) cells) was injected directly into the contusion epicenter at a depth of 1 mm using a Hamilton syringe positioned with a stereotaxic instrument (RWD Life Science, China) and driven by a syringe pump (KDS LEGATO 130, KD Scientific, USA) at a flow rate of 1 µl/min.
Three days of exercise was implemented in all groups prior to SCI injury for acclimatization. Treadmill training began 3 days after SCI. The mice in the TMT and BMMSCs + TMT group were allowed to run on the treadmill apparatus (ZH-PT, China) for 8 weeks (20 min/day, 6 days per week). The training speed was started at 4 m/min and gradually increased according to the tolerance of the mice (the maximum speed was 9 m/min).
Locomotor recovery was evaluated by the Basso Mouse Scale (BMS), horizontal ladder test, and footprint analysis [35–37].
The BMS was assessed by two observers who were blinded to the experimental treatments before injury, 1 day after the injury, and then weekly after injury in an open field. The BMS was mainly used to determine the particular behavioral components of locomotion, such as trunk stability, plantar stepping, coordination, and paw position.
The horizontal ladder test requires adequate sensorimotor function to contact and feel the ladder rungs when stepping; thus, it is more sensitive to locomotor deficits [38]. The ladder consisted of 30 metal rungs (2 mm in diameter) spaced regularly (1.3 cm apart) measuring 30 cm in height. Mice were exposed to the horizontal ladder at least once prior to surgery for acclimatization to the testing procedure. The walking ability of each mouse across the 30 metal rungs was recorded by video during each testing period. The hindlimb error rate (wrong steps/total steps) was evaluated weekly by a blinded investigator. A correct step corresponded to plantar or toe placements on rungs without slipping or dragging.
For the footprint analysis, mouse forepaws and hindlimbs were painted with red and black dyes, respectively. Mice were then allowed to walk in a straight path lined with white paper.
T2-weighted sagittal magnetic resonance images were acquired using a 7T small animal MR system, Biospec USR70/30 (Bruker Corporation, Karlsruhe, Germany). During MRI, the mice were anesthetized by inhalation of 3% isoflurane and maintained under anesthesia by inhalation of 1.5% isoflurane through a face mask. The parameters were as follows: repetition time (TR) = 3000 ms, echo time (TE) = 27 ms, field of view = 40 x 30 mm, number of averages = 2, matrix size = 256 x 256, slice number = 10, slice thickness = 0.8 mm, and slice gap = 0 mm. Six animals were observed for each group. The lesion area was measured manually using ImageJ software in each slice, and lesion volumes were calculated by summing lesion areas in each slice multiplied by the thickness of the slice.
Eight weeks after intervention, the animals were deeply anesthetized with isoflurane and transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Spinal cords (1 cm long for longitudinal sections and 0.5 cm long for transverse sections) centered at the injury epicenter were dissected and fixed in 4% neutral buffered paraformaldehyde for 24 h, embedded in paraffin and cut into 2.5-µm-thick slices. Longitudinal sections of the injured spinal cord were stained with Masson's trichrome to assess scar formation and with hematoxylin and eosin (H&E) for morphological detection (n = 3 per group). Transverse sections were stained with Nissl (cresyl violet) to count neurons after injury (n = 3 per group).
Spinal cord tissue samples were collected, and transverse sections of the spinal cord were fixed in 2.5% glutaraldehyde in 0.1 M PBS at 4°C for 24 h. Then, samples were postfixed in 1% osmium tetroxide in PBS, dehydrated in graded alcohols, and embedded in epoxy resin (Epon 812). Ultrathin sections of 50 nm were stained with uranyl acetate and lead citrate and finally examined with a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan).
The animals were sacrificed 8 weeks following injury, and spinal cord tissue at the injury epicenter was harvested for western blot analysis with n = 3 mice per group. Homogenates from spinal cord tissue were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitor cocktails at 4°C. Protein concentrations were determined by using a bicinchoninic acid (BCA) kit (Beyotime). After normalization, samples were run on 10% SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked for 1 hour at room temperature and incubated with a specific primary antibody at 4°C overnight. After washing with Tris-buffered saline plus Tween (TBST), the membranes were incubated with anti-rabbit immunoglobulin G (IgG) horseradish-peroxidase-conjugated secondary antibodies for 1 h. Immunoreactive bands were visualized using an enhanced chemiluminescent detection system (Bio–Rad, USA). The immunoreactive bands were quantified by densitometric analysis using ImageJ Software. The primary antibodies used in this study included glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), neuronal nuclei (NeuN), neurofilament-200 (NF200), postsynaptic density protein (PSD), synaptophysin (SYN), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and p-mTOR, mTOR, and PTEN antibodies (all from Proteintech, China), AKT (Protein kinase B), p-AKT, PI3K (Phosphatidylinositol 3-kinase), and p-PI3K antibodies (all from Cell Signaling Technology, USA), and nerve growth factor (NGF) antibody (from Abcam, USA).
Spinal cord tissue was prepared as described previously and sectioned in the longitudinal or transverse plane at a thickness of 2.5 µm. Sections were deparaffinized and rehydrated with xylene 2 x 10 min, 100% ethanol 2 x 5 min, and 96% ethanol, 80% ethanol, 70% ethanol, and H2O for 5 min each. Antigen retrieval was performed with citrate buffer (pH 6.2), and the sections were blocked with 10% goat serum. Sections were then incubated at 4°C overnight with the following primary antibodies: GFAP, MBP, NeuN, NF200, PSD, and SYN antibodies (all from Proteintech, China) and NGF antibody (from Abcam, USA). After washing, sections were then stained for 1 h at 37°C with the following fluorescent secondary antibodies: fluorescein isothiocyanate (FITC)-conjugated and rhodamine (TRITC)-conjugated secondary antibody (Proteintech, China). Nuclei were visualized by staining with DAPI. Images were acquired using an N-STORM & A1 (Nikon, Tokyo, Japan) microscope.
Data were expressed as the means ± SEM. Statistical analysis was performed using Prism 8.0 (GraphPad Software Inc., San Diego, CA). BMS scores and hindlimb error rates were analyzed using two-way analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons. All other experiments were analyzed with one-way ANOVA followed by Tukey's post hoc multiple comparison test. A P-value ≤ 0.05 was considered significant.
After 3 passages, the cell surface markers of BMMSCs were determined by flow cytometry. The results showed positive expression (> 99%) of CD29 and CD44 and negative expression (< 1%) of CD34 and CD45, which indicated the high purity of the extracted BMMSCs (Additional file 1: Figure S1).
BMS score assessment, and horizontal ladder and footprint analyses were then performed to evaluate the functional recovery of mice subjected to the different treatments. On the first day after injury, BMS assessment showed that the hindlimbs of all mice were completely paralyzed (BMS score = 0), and then the mice showed different extents of recovery (Fig. 1A). Starting at 3 weeks after injury, the extent of functional recovery began to reveal the differences between the control group and the treatment groups (TMT, BMMSCs, BMMSCs + TMT; p < 0.001, p < 0.001, p < 0.001, respectively). This difference remained statistically significant up to 8 weeks after SCI. The significant difference between the BMMSCs group and the BMMSCs + TMT group emerged 6 weeks after SCI and persisted until the end of the trial (p < 0.05). In addition, a statistically significant difference appeared at 7 weeks and persisted at 8 weeks after injury between the TMT group and the BMMSCs + TMT group (p < 0.05, p < 0.05, respectively). The footprint test intuitively reflected differences in the tracks of hindlimbs among different groups. Mice in the control group showed obvious dragging of hindlimbs (blue ink) compared with the clear footprints of mice in the sham group, whereas mice in the BMMSCs + TMT group displayed fairly consistent hindlimb track with a few stumbled walking trajectories at 8 weeks after SCI (Fig. 1B). Animals in the TMT or BMMSCs group partially recovered coordination of the front and hind limbs but still showed partial drag. We also measured fine differences in motor function using a horizontal ladder test (Fig. 1C). Over an 8-week testing period, all mice subjected to SCI showed a progressive decrease in the error rates (Fig. 1D). The mean error rate was significantly lower in the BMMSCs + TMT group than in the control group at 4 weeks (p < 0.05) after injury, which persisted until 8 weeks after injury (p < 0.01). However, no difference was found between the TMT or BMMSCs alone group and the control group at any time point examined. Taken together, these results show that the combined intervention can better promote locomotor function recovery than individual therapy after SCI.
We investigated the effect of the combined therapy on the structural repair of the injured spinal cord tissue. The lesion volume of the injured spinal cord was measured at 8 weeks after injury using T2-weighted MRI (Fig. 2A). Compared with that in the control group, a significant reduction in lesion volume was found in mice that received treatment (TMT, BMMSCs, BMMSCs + TMT; p < 0.01, p < 0.001, p < 0.001, respectively) (Fig. 2C). Nevertheless, the differences between the combined treatment and TMT or BMMSCs alone were not statistically significant. The histological morphology changes in the injured spinal cord at 8 weeks after injury were evaluated using H&E staining (Fig. 2B). Consistent with our MRI results, the relative lesion area in the injured spinal cords treated with TMT, BMMSCs, and BMMSCs + TMT was significantly reduced (p < 0.001, p < 0.001, p < 0.001, respectively), with less damaged tissue than that in the control group (Fig. 2D). Masson's trichrome staining was also used to estimate scar formation at the site of injury (Fig. 2B). TMT or BMMSCs alone did not significantly affect the fibrotic area, whereas a significantly lower average percentage of fibrosis was observed in the BMMSCs + TMT group than in the control group (42.57 ± 9.69% vs. 100.00 ± 7.82%, p < 0.001) (Fig. 2E). These results suggest that TMT or BMMSCs alone can promote the preservation of spinal cord tissue, but no significant synergism was noted between them. Furthermore, only the combination of BMMSCs and TMT can result in a significant reduction in scar formation.
To determine the neuroprotective effect of intervention on SCI mice, we examined neuronal density in the anterior horn of the injured spinal cord at 8 weeks following SCI by double immunofluorescent staining for GFAP (an astrocyte marker) and NeuN (a neuronal marker) (Fig. 3A). SCI induced a significant decrease in NeuN-positive neurons in the anterior horn of the injured spinal cord. The immunofluorescence results showed that the relative density of NeuN in the BMMSCs + TMT group significantly increased compared with those in the control, TMT alone, and BMMSCs alone groups (p < 0.001, p < 0.05, p < 0.05, Fig. 3C). However, TMT or BMMSCs alone did not significantly affect neuronal viability. We also investigated whether the combination treatment resulted in enhanced production of nerve growth factor (NGF), which is a crucial neurotrophin for neuronal growth and survival (Fig. 3B). TMT or BMMSCs alone did not provide any obvious beneficial effects on NGF production. Only when the two strategies were combined was the NGF level at the injured epicenter region significantly increased compared with that in the control group (p < 0.001, Fig. 3D). For Nissl staining, as shown in Fig. 3E, a large number of normal neurons in the sham group displayed an integrative and granular-like morphology, while in the control group, their numbers were significantly decreased, and most of them exhibited shrunken cell bodies and Nissl granule dissolution. A quantitative comparison of Nissl bodies showed that only the BMMSCs + TMT group exhibited a significant increase compared with the control group. in the number of surviving neurons (p < 0.05, Fig. 3F). Additionally, western blot analysis of the NeuN and NGF proteins further confirmed the above results (Fig. 3G, 3H, 3I). We also detected the protein levels of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF), but the differences among all groups were not statistically significant (Figure S2A, S2C, S2D). These observations indicated that the combination therapy could exert a protective effect on neurons and promote the repair of damaged tissue after SCI.
The capacity of axon regeneration at the lesion center of the spinal cord was evaluated by double immunofluorescent staining for GFAP and NF200 (an axonal marker) (Fig. 4A, 4B). GFAP-positive astrocytes accumulated along the lesion border (Fig. 4A). Notably, more NF200-positive, injured axons crossed the lesion border in the BMMSCs + TMT group compared with the axons observed in the TMT or BMMSCs alone group and the control group (p < 0.05, p < 0.05, p < 0.001, respectively) (Fig. 4E). In addition, the expression of NF200 in the TMT or BMMSCs group was significantly increased compared with that in the control group (p < 0.05). Double immunofluorescent staining for GFAP and MBP (a myelin marker) was performed to determine the effect of the intervention on remyelination (Fig. 4C). We also detected the microstructure of myelin using TEM (Fig. 4D). The immunofluorescence results suggested that TMT or BMMSCs alone increased the expression of MBP (p < 0.05), but the combination of the two strategies did not result in superior improvement compared with TMT or BMMSCs alone (Fig. 4F). Furthermore, TEM analysis revealed that only the BMMSCs + TMT group had significantly increased myelin thickness, as quantified by the G-ratio (p < 0.001, BMMSCs + TMT versus control, Fig. 4G). Moreover, western blot results further confirmed the immunofluorescence findings of NF200 and MBP (Fig. 5H, 5I). Overall, these data indicate that the combination therapy can exert a more obvious effect on axon regeneration and remyelination than TMT or BMMSCs alone.
Immunofluorescence staining of PSD (a presynaptic marker protein) and SYN (a postsynaptic marker protein) in the injured spinal cord was performed to evaluate synaptic function (Fig. 5A, 5B). The expression levels of PSD and SYN at the injured epicenter region were substantially increased in the BMMSCs + TMT group compared with the control group (p < 0.001; p < 0.01). TMT or BMMSCs alone tended to increase the expression levels of PSD and SYN, but no significant difference was observed between the TMT or BMMSC alone group and the control group (Fig. 5C, 5D). Western blot analysis for PSD and SYN (Fig. 5E, 5F, 5G) also confirmed the above results, indicating that TMT and BMMSCs acted synergistically to enhance synaptic plasticity better than the individual interventions.
Studies have confirmed that the PI3K/AKT/mTOR signaling pathway is involved in the growth of central nervous system (CNS) axons during development [39, 40]. During CNS maturity, phosphatase and tensin homolog on chromosome 10 (PTEN) suppresses the activity of this signaling pathway, accounting for the failure of axon regeneration in the injured spinal cord[41]. To further determine whether the combinatorial therapies promote SCI recovery by targeted activation of the PI3K/AKT/mTOR pathway, we performed western blot analysis to detect the protein levels of the key molecules in this pathway (Fig. 6A). The results indicate that only the combination therapy significantly enhanced the expression of p-PI3K, p-AKT and p-mTOR compared with the control group (p < 0.05; p < 0.05; p < 0.001, Fig. 6B, 6C, 6D). Silencing of PTEN, a negative regulator of mTOR, was shown to promote axon growth in animal models after SCI. In our study, although the expression level of PTEN in the BMMSCs + TMT group tended to slightly decrease compared with that in the control group, the differences were not statistically significant (p > 0.05, Figure S2A, S2B). The above data revealed that BMMSCs implantation combined with TMT promoted axonal regeneration and neuroplasticity via the PI3K/AKT/mTOR signaling pathway.
Although many therapeutic interventions have shown promise in treating SCI, focusing on a single aspect of repair cannot facilitate successful and functional regeneration in patients following SCI. Therefore, a combination of various interventions addressing the multiple aspects of SCI pathology is likely required. In this study, we opted for the combinatorial approach of neuroprotection and rehabilitation, capitalizing on cell transplantation and functional sensorimotor training to promote nerve regeneration and functional recovery. Treatment targets for SCI that can improve functional recovery include reduction of secondary damage, replacement of lost cells, removal of inhibitory molecules, axon regeneration through targeting neuronal mechanisms, resupply of trophic support, remyelination of demyelinated axons and rehabilitation for circuit remodeling [42–44]. Thus, in the present study, multiple integrated evidence derived in vivo was performed to assess SCI recovery, including locomotor performance, histopathological lesions, scar formation, axon growth and synapse remodeling and myelin regeneration.
Our results indicated that the combined treatment with BMMSCs and TMT showed the best therapeutic effect on functional recovery compared with other groups. The enhanced motor functional recovery by the combination therapy can probably be explained as follows. The combination of BMMSCs and TMT markedly reduced fibrotic scar tissue, protected neurons, promoted remyelination and axonal generation, and increased synapse formation, all to a larger extent than either TMT or BMMSCs alone. More strikingly, although the BMS score of each single therapy was significantly higher than that of the control group at the end of the trial, the mean error rate of hindlimbs between TMT or BMMSCs alone and the control group was not statistically significant. A possible explanation is that stepping across the rungs requires precise foot placement and grasping, which may provide a challenge to mice with poor locomotor performance [45, 46]. Additionally, we noted that both single therapy and the combined therapy greatly reduced the tissue damage assessed by MRI and H&E staining, and the combined therapy did not obviously enhance these independent effects of each single therapy. Thus, physical exercise or cell transplantation alone can be reasonably considered to also be able to promote tissue preservation.
Stem cell transplantation can promote SCI repair and functional improvement by differentiating into neurons or glial cells to replace damaged cells and secreting a variety of neurotrophic factors to protect the injured tissue and enhance axon regeneration [47, 48]. BMMSCs are generally accepted to have the advantages of high biosafety, wide biological effects and low immunogenicity [49]. In this study, we also observed that BMMSCs can promote axon and myelin regeneration, which is similar to findings in previous studies [13, 47]. Scar formation is a pivotal determinant in limiting axonal regeneration after SCI [50]. Reactive astrogliosis is typically associated with the formation of compact scar borders around the inflammatory core [51]. Moreover, border-forming astrocytes increase the deposition of chondroitin sulfate proteoglycans (CSPGs), the major matrix contents of the glial scar, which may pose a physical and chemical barrier to axon outgrowth [52–55]. Our present data have shown that only the BMMSCs + TMT treatment can lead to a substantial decrease in scar formation, which may provide a significant contribution to axon regeneration in the injured spinal cord. Indeed, we found that the combination of BMMSCs and TMT led to the highest expression of neurofilaments in the injured spinal cord.
SCI leads to the disruption of neural connectivity, thus resulting in severe permanent neurological disability. Restoration of function relies on promoting the formation of new connections and circuits [55, 56]. The remodeling of functional neural circuits in the spinal cord and brain may need to be driven by rehabilitation [57]. Combined treatments targeting the promotion of neuronal plasticity seem to be an effective approach. Current reports on the role of cell transplantation with exercise training are limited to several preclinical studies. A study in rats reported no evidence of functional recovery after bone marrow stromal cell transplantation or physical exercise alone or after both treatments [58]. In a subsequent chronic SCI mouse study, neural stem cell transplantation combined with TMT treatment was shown to significantly enhance functional recovery and facilitate neuronal differentiation of transplanted cells compared with either treatment alone [59]. Another recent study reported the functional and morphological benefits of a combinatorial approach with BMMSCs and early TMT treatment in a compression SCI mouse model [60]. Therefore, further investigations still need to explore the detailed mechanisms.
An intriguing finding in our study is that the BMMSCs + TMT group remarkably upregulated the PI3K/Akt/mTOR pathway. Mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, can stimulate ribosomal protein translation [61]. Recent research has suggested that axonal growth in the injured CNS is mediated through PI3K/AKT/mTOR signalling pathway [62, 63]. This pathway is suppressed in the injured CNS, which may limit the protein synthesis necessary for axon regeneration [64]. PTEN is considered to be an essential factor that can negatively regulate the PI3K/AKT/mTOR pathway, and genetic deletion of this molecule has been shown to increase the intrinsic growth capacity of neurons [65]. Phosphatidylinositol 3-kinases (PI3Ks) are a class of lipid kinases that convert phosphatidylinositol (4,5)-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and then activate AKT and mTOR, ultimately mediating neuroprotection and axogenic protein synthesis [66]. After SCI, the upregulation of PTEN can restrict the binding of AKTs to membranes by dephosphorylating PIP3 to PIP2, leading to the inactivation of the PI3K/AKT/mTOR pathway [40]. In our study, we also found that the expression of PTEN was upregulated in spinal cord tissues derived from mice subjected to SCI. We hypothesized that our interventions would reduce the expression of PTEN and then promote the activation of the PI3K/AKT/mTOR pathway. The results indicated that the combination group tended to downregulate the expression of PTEN, but the differences were not significant (Additional file 2: Figure S2A, S2B). One speculation is that the combinatorial approach of BMMSCs transplantation and exercise training may not only target PTEN to regulate the PI3K/AKT/mTOR signaling pathway. Notably, neurotrophic factors are crucial for supporting the viability of neurons and the growth of axons during mammalian CNS development [67]. Furthermore, the binding of neurotrophic factors to tyrosine kinase receptors (Trk) triggers their dimerization and autophosphorylation of tyrosine residues within the intracellular kinase domain, which can activate the PI3K/AKT/mTOR intracellular signaling pathway [40]. As expected, the NGF level at the lesion center was significantly increased in the BMMSCs + TMT group (Fig. 3B, 3D, 3G, 3I). Based on these findings, we speculated that BMMSCs combined with TMT exerts a neuroprotective effect by elevating NGF levels and then activating PI3K/AKT/mTOR signaling. Additionally, BDNF and VEGF levels were not obviously altered by either single therapy or combination therapy in this study (Figure S2A, S2C, S2D). However, previous studies demonstrated that both BMMSCs and TMT have the potential to increase BDNF levels in the injured spinal cord [59, 60]. This discrepancy might be due to different time points being examined. Further studies will be required to identify the complex interactions between exercise training and cell transplantation and determine the best temporal window.
In summary, the findings of the present study indicate that BMMSCs translation combined with exercise training synergistically improved motor function in paralyzed hindlimbs and established favorable conditions for functional restoration after SCI from the following aspects: promoting axonal regeneration and remyelination, enhancing synaptic plasticity and neurotrophin secretion, suppressing scar formation, and protecting neurons. Taken together, this study presents a promising combinatorial therapy for patients suffering from SCI. In addition, further studies are required to identify and understand the complex interactions between therapies.
SCI: spinal cord injury, BMMSCs: bone marrow mesenchymal stem cells, TMT: treadmill training, BMS: Basso Mouse Scale, MRI: Magnetic resonance imaging, H&E: hematoxylin and eosin, TEM: Transmission electron microscopy, GFAP: glial fibrillary acidic protein, MBP: myelin basic protein, NeuN: neuronal nuclei, NF200: neurofilament-200, PSD: postsynaptic density protein, SYN: synaptophysin, BDNF: brain-derived neurotrophic factor, VEGF: vascular endothelial growth factor, NGF: nerve growth factor, PTEN: phosphatase and tensin homolog on chromosome 10, mTOR: Mammalian target of rapamycin, AKT: Protein kinase B, PI3K: Phosphatidylinositol 3-kinase.
Acknowledgments
None.
Funding
This work was supported by the National Natural Science Foundation of China [Grant No. 81572231], and the Project of the Science and Technology Department in Sichuan province [Grant No. 2019YJ0119].
Authors’ Contributions
X.S. and Q.W. conceived the idea and designed the research studies. X.S. and L.H. conducted the experiments. C.F., L.L., L.W., and G.P. participated in SCI model preparation and data analysis. Y.W., Q.Z., and H.C. acquired and analyzed the data. X.S. drafted the initial manuscript. Q.W. and C.H. reviewed and revised the manuscript. All authors approved the final version of manuscript.
Availability of data and materials
All data generated during this study are included in this article.
Ethics approval and consent to participate
All procedures were approved by the Laboratory Animal Ethics Committee of the West China Hospital of Sichuan University (Ethical approval number: 20211198A).
Consent for publication
Not applicable.
Competing interests
All authors declare no competing interests.