Mesenchymal stem cells and treadmill training enhance function and promote tissue preservation after spinal cord injury.

Spinal cord injury (SCI) is considered a serious neurological disorder that can lead to severe sensory, motor and autonomic deficits. In this work, we investigated whether cell therapy associated with physical activity after mouse SCI could promote morphological and functional outcomes, using a lesion model established by our group. Mesenchymal stem cells (8 × 105 cells/2 µL) or DMEM (2 µL), were injected in the epicenter of the lesion at 7 days after SCI, and the mice started a moderate treadmill training 14 days after injury. Functional assessments were performed weekly up to 8 weeks after injury when the morphological analyses were also performed. Four injured groups were analyzed: DMEM (SCI plus DMEM injection), MSCT (SCI plus MSC injection), DMEM + TMT (SCI plus DMEM injection and treadmill training) and MSCT + TMT (SCI plus MSC injection and treadmill training). The animals that received the combined therapy (MSCT + TMT) were able to recover and maintained the better functional results throughout the analyzed period. The morphometric analysis from MSCT + TMT group evidenced a larger spared white matter area and a higher number of preserved myelinated fibers with the majority of them reaching the ideal G-ratio values, when compared to other groups. Ultrastructural analysis from this group, using transmission electron microscopy, showed better tissue preservation with few microcavitations and degenerating nerve fibers. Also, this group exhibited a significantly higher neurotrophin 4 (NT4) expression as compared to the other groups. The results provided by this study support the conclusion that the association of strategies is a potential therapeutic approach to treat SCI, with the possibility of translation into the clinical practice.

Injection of bone marrow mesenchymal stem cells by intravenous or intraperitoneal routes is a viable alternative to spinal cord injury treatment in mice.

In spite of advances in surgical care and rehabilitation, the consequences of spinal cord injury (SCI) are still challenging. Several experimental therapeutic strategies have been studied in the SCI field, and recent advances have led to the development of therapies that may act on the inhibitory microenvironment. Assorted lineages of stem cells are considered a good treatment for SCI. This study investigated the effect of systemic transplantation of mesenchymal stem cells (MSCs) in a compressive SCI model. Here we present results of the intraperitoneal route, which has not been used previously for MSC administration after compressive SCI. We used adult female C57BL/6 mice that underwent laminectomy at the T9 level, followed by spinal cord compression for 1 minute with a 30-g vascular clip. The animals were divided into five groups: sham (anesthesia and laminectomy but without compression injury induction), MSC i.p. (intraperitoneal injection of 8 × 105 MSCs in 500 µL of DMEM at 7 days after SCI), MSC i.v. (intravenous injection of 8 × 105 MSCs in 500 µL of DMEM at 7 days after SCI), DMEM i.p. (intraperitoneal injection of 500 µL of DMEM at 7 days after SCI), DMEM i.v. (intravenous injection of 500 µL of DMEM at 7 days after SCI). The effects of MSCs transplantation in white matter sparing were analyzed by luxol fast blue staining. The number of preserved fibers was counted in semithin sections stained with toluidine blue and the presence of trophic factors was analyzed by immunohistochemistry. In addition, we analyzed the locomotor performance with Basso Mouse Scale and Global Mobility Test. Our results showed white matter preservation and a larger number of preserved fibers in the MSC groups than in the DMEM groups. Furthermore, the MSC groups had higher levels of trophic factors (brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and neurotrophin-4) in the spinal cord and improved locomotor performance. Our results indicate that injection of MSCs by either intraperitoneal or intravenous routes results in beneficial outcomes and can be elected as a choice for SCI treatment.

Blockade of ATP P2X7 receptor enhances ischiatic nerve regeneration in mice following a crush injury.

Preventing damage caused by nerve degeneration is a great challenge. There is a growing body of evidence implicating extracellular nucleotides and their P2 receptors in many pathophysiological mechanisms. In this work we aimed to investigate the effects of the administration of Brilliant Blue G (BBG) and Pyridoxalphosphate-6-azophenyl-2', 4'- disulphonic acid (PPADS), P2X7 and P2 non-selective receptor antagonists, respectively, on sciatic nerve regeneration. Four groups of mice that underwent nerve crush lesion were used: two control groups treated with vehicle (saline), a group treated with BBG and a group treated with PPADS during 28days. Gastrocnemius muscle weight was evaluated. For functional evaluation we used the Sciatic Functional Index (SFI) and the horizontal ladder walking test. Nerves, dorsal root ganglia and spinal cords were processed for light and electron microscopy. Antinoceptive effects of BBG and PPADS were evaluated through von Frey E, and the levels of IL-1ß and TNF-α were analyzed by ELISA. BBG promoted an increase in the number of myelinated fibers and on axon, fiber and myelin areas. BBG and PPADS led to an increase of TNF-α and IL-1ß in the nerve on day 1 and PPADS caused a decrease of IL-1ß on day 7. Mechanical allodynia was reversed on day 7 in the groups treated with BBG and PPADS. We concluded that BBG promoted a better morphological regeneration after ischiatic crush injury, but this was not followed by anticipation of functional improvement. In addition, both PPADS and BBG presented anti-inflammatory as well as antinociceptive effects.

Chronic spinal cord lesions respond positively to tranplants of mesenchymal stem cells.

PURPOSE: Despite substantial advances in surgical care and rehabilitation, the consequences of spinal cord injury (SCI) continue to present major challenges. Here we investigate whether transplantation of mesenchymal stem cells (MSCs) in mice during the chronic stage of SCI has benefits in terms of morphological and functional outcomes. METHODS: Mice were subjected to laminectomy at the T9 level, followed by a 1 minute spinal cord compression with a vascular clip. Four weeks later, 8 × 105 MSCs obtained from GFP mice were injected into the injury site. After eight weeks the analyses were performed. RESULTS: The spinal cords of MSC-treated animals exhibited better white-matter preservation, greater numbers of fibers, higher levels of trophic factor expression, and better ultrastructural tissue organization. Furthermore, transplanted MSCs were not immunoreactive for neural markers, indicating that these cells mediate functional recovery through a paracrine effect, rather than by transforming into and replacing damaged glia in the spinal cord. MSC-treated mice also showed better functional improvement than control animals. CONCLUSION: We conclude that MSC-based cell therapy, even when applied during the chronic phase of SCI, leads to changes in a number of structural and functional parameters, all of which indicate improved recovery.

A combination of Schwann-cell grafts and aerobic exercise enhances sciatic nerve regeneration.

BACKGROUND: Despite the regenerative potential of the peripheral nervous system, severe nerve lesions lead to loss of target-organ innervation, making complete functional recovery a challenge. Few studies have given attention to combining different approaches in order to accelerate the regenerative process. OBJECTIVE: Test the effectiveness of combining Schwann-cells transplantation into a biodegradable conduit, with treadmill training as a therapeutic strategy to improve the outcome of repair after mouse nerve injury. METHODS: Sciatic nerve transection was performed in adult C57BL/6 mice; the proximal and distal stumps of the nerve were sutured into the conduit. Four groups were analyzed: acellular grafts (DMEM group), Schwann cell grafts (3×105/2 µL; SC group), treadmill training (TMT group), and treadmill training and Schwann cell grafts (TMT + SC group). Locomotor function was assessed weekly by Sciatic Function Index and Global Mobility Test. Animals were anesthetized after eight weeks and dissected for morphological analysis. RESULTS: Combined therapies improved nerve regeneration, and increased the number of myelinated fibers and myelin area compared to the DMEM group. Motor recovery was accelerated in the TMT + SC group, which showed significantly better values in sciatic function index and in global mobility test than in the other groups. The TMT + SC group showed increased levels of trophic-factor expression compared to DMEM, contributing to the better functional outcome observed in the former group. The number of neurons in L4 segments was significantly higher in the SC and TMT + SC groups when compared to DMEM group. Counts of dorsal root ganglion sensory neurons revealed that TMT group had a significant increased number of neurons compared to DMEM group, while the SC and TMT + SC groups had a slight but not significant increase in the total number of motor neurons. CONCLUSION: These data provide evidence that this combination of therapeutic strategies can significantly improve functional and morphological recovery after sciatic injury.

Mesenchymal stem cells in a polycaprolactone conduit promote sciatic nerve regeneration and sensory neuron survival after nerve injury.

Despite the fact that the peripheral nervous system is able to regenerate after traumatic injury, the functional outcomes following damage are limited and poor. Bone marrow mesenchymal stem cells (MSCs) are multipotent cells that have been used in studies of peripheral nerve regeneration and have yielded promising results. The aim of this study was to evaluate sciatic nerve regeneration and neuronal survival in mice after nerve transection followed by MSC treatment into a polycaprolactone (PCL) nerve guide. The left sciatic nerve of C57BL/6 mice was transected and the nerve stumps were placed into a biodegradable PCL tube leaving a 3-mm gap between them; the tube was filled with MSCs obtained from GFP+ animals (MSC-treated group) or with a culture medium (Dulbecco's modified Eagle's medium group). Motor function was analyzed according to the sciatic functional index (SFI). After 6 weeks, animals were euthanized, and the regenerated sciatic nerve, the dorsal root ganglion (DRG), the spinal cord, and the gastrocnemius muscle were collected and processed for light and electron microscopy. A quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers in the group that received, within the nerve guide, stem cells. The number of neurons in the DRG was significantly higher in the MSC-treated group, while there was no difference in the number of motor neurons in the spinal cord. We also found higher values of trophic factors expression in MSC-treated groups, especially a nerve growth factor. The SFI revealed a significant improvement in the MSC-treated group. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase enzyme, suggesting an improvement of reinnervation and activity in animals that received MSCs. Immunohistochemistry documented that some GFP+ -transplanted cells assumed a Schwann-cell-like phenotype, as evidenced by their expression of the S-100 protein, a Schwann cell marker. Our findings suggest that using a PCL tube filled with MSCs is a good strategy to improve nerve regeneration after a nerve transection in mice.

Transplantation of bone-marrow-derived cells into a nerve guide resulted in transdifferentiation into Schwann cells and effective regeneration of transected mouse sciatic nerve.

Peripheral nerves possess the capacity of self-regeneration after traumatic injury. Nevertheless, the functional outcome after peripheral-nerve regeneration is often poor, especially if the nerve injuries occur far from their targets. Aiming to optimize axon regeneration, we grafted bone-marrow-derived cells (BMDCs) into a collagen-tube nerve guide after transection of the mouse sciatic nerve. The control group received only the culture medium. Motor function was tested at 2, 4, and 6 weeks after surgery, using the sciatic functional index (SFI), and showed that functional recovery was significantly improved in animals that received the cell grafts. After 6 weeks, the mice were anesthetized, perfused transcardially, and the sciatic nerves were dissected and processed for transmission electron microscopy and light microscopy. The proximal and distal segments of the nerves were compared, to address the question of improvement in growth rate; the results revealed a maintenance and increase of nerve regeneration for both myelinated and non-myelinated fibers in distal segments of the experimental group. Also, quantitative analysis of the distal region of the regenerating nerves showed that the numbers of myelinated fibers, Schwann cells (SCs) and g-ratio were significantly increased in the experimental group compared to the control group. The transdifferentiation of BMDCs into Schwann cells was confirmed by double labeling with S100/and Hoechst staining. Our data suggest that BMDCs transplanted into a nerve guide can differentiate into SCs, and improve the growth rate of nerve fibers and motor function in a transected sciatic-nerve model.

Predifferentiated embryonic stem cells promote functional recovery after spinal cord compressive injury.

We tested the effects of mouse embryonic stem cells (mES) grafts in mice spinal cord injury (SCI). Young adult female C57/Bl6 mice were subjected to laminectomy at T9 and 1-minute compression of the spinal cord with a vascular clip. Four groups were analyzed: laminectomy (Sham), injured (SCI), vehicle (DMEM), and mES-treated (EST). mES pre-differentiated with retinoic acid were injected (8 x 10(5) cells/2 microl) into the lesion epicenter, 10 min after SCI. Basso mouse scale (BMS) and Global mobility test (GMT) were assessed weekly up to 8 weeks, when morphological analyses were performed. GMT analysis showed that EST animals moved faster (10.73+/-0.9076, +/-SEM) than SCI (5.581+/-0.2905) and DMEM (5.705+/-0.2848), but slower than Sham animals (15.80+/-0.3887, p<0.001). By BMS, EST animals reached the final phase of locomotor recovery (3.872+/-0.7112, p<0.01), while animals of the SCI and DMEM groups improved to an intermediate phase (2.037+/-0.3994 and 2.111+/-0.3889, respectively). White matter area and number of myelinated nerve fibers were greater in EST (46.80+/-1.24 and 279.4+/-16.33, respectively) than the SCI group (39.97+/-0.925 and 81.39+/-8.078, p<0.05, respectively). EST group also presented better G-ratio values when compared with SCI group (p<0.001). Immunohistochemical revealed the differentiation of transplanted cells into astrocytes, oligodendrocytes, and Schwann cells, indicating an integration of transplanted cells with host tissue. Ultrastructural analysis showed, in the EST group, better tissue preservation and more remyelination by oligodendrocytes and Schwann cells than the other groups. Our results indicate that acute transplantation of predifferentiated mES into the injured spinal cord increased the spared white matter and number of nerve fibers, improving locomotor function.