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 . 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 . Reactive astrogliosis is typically associated with the formation of compact scar borders around the inflammatory core . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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.