Molecular approaches for spinal cord injury treatment.

Injuries to the spinal cord result in permanent disabilities that limit daily life activities. The main reasons for these poor outcomes are the limited regenerative capacity of central neurons and the inhibitory milieu that is established upon traumatic injuries. Despite decades of research, there is still no efficient treatment for spinal cord injury. Many strategies are tested in preclinical studies that focus on ameliorating the functional outcomes after spinal cord injury. Among these, molecular compounds are currently being used for neurological recovery, with promising results. These molecules target the axon collapsed growth cone, the inhibitory microenvironment, the survival of neurons and glial cells, and the re-establishment of lost connections. In this review we focused on molecules that are being used, either in preclinical or clinical studies, to treat spinal cord injuries, such as drugs, growth and neurotrophic factors, enzymes, and purines. The mechanisms of action of these molecules are discussed, considering traumatic spinal cord injury in rodents and humans.

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.

A highly reproducible mouse model of compression spinal cord injury.

Experimental spinal cord injury (SCI) can maintain the continuity of the spinal cord, as in the contusion (e.g., weight-fall) or compression models, or not, when there is a partial or a complete transection. The majority of acute human SCI is not followed by complete transection, but there is a combination of contusion, compression, and possibly partial transection. The method described here is a compressive mouse model that presents a combination of contusion and compression components and has many facilities in its execution. This lesion was established by our group and represents a simple, reliable, and inexpensive clip compression model with functional and morphological reproducibility. In this chapter we describe, step by step, the protocol of this experimental SCI.

Human dental pulp cells: a new source of cell therapy in a mouse model of compressive spinal cord injury.

Strategies aimed at improving spinal cord regeneration after trauma are still challenging neurologists and neuroscientists throughout the world. Many cell-based therapies have been tested, with limited success in terms of functional outcome. In this study, we investigated the effects of human dental pulp cells (HDPCs) in a mouse model of compressive spinal cord injury (SCI). These cells present some advantages, such as the ease of the extraction process, and expression of trophic factors and embryonic markers from both ecto-mesenchymal and mesenchymal components. Young adult female C57/BL6 mice were subjected to laminectomy at T9 and compression of the spinal cord with a vascular clip for 1 min. The cells were transplanted 7 days or 28 days after the lesion, in order to compare the recovery when treatment is applied in a subacute or chronic phase. We performed quantitative analyses of white-matter preservation, trophic-factor expression and quantification, and ultrastructural and functional analysis. Our results for the HDPC-transplanted animals showed better white-matter preservation than the DMEM groups, higher levels of trophic-factor expression in the tissue, better tissue organization, and the presence of many axons being myelinated by either Schwann cells or oligodendrocytes, in addition to the presence of some healthy-appearing intact neurons with synapse contacts on their cell bodies. We also demonstrated that HDPCs were able to express some glial markers such as GFAP and S-100. The functional analysis also showed locomotor improvement in these animals. Based on these findings, we propose that HDPCs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans.

Effects of stretching on morphological and biochemical aspects of the extracellular matrix of the rat calcaneal tendon.

Several studies have demonstrated the relationship between exercise and the extracellular matrix of muscle tendons, and have described alterations in their structural and biochemical properties when subjected to strenuous exercise. However, little is known about what happens to tendons when they are subjected to stretching. We evaluated the changes in the composition and structure of rat calcaneal tendons subjected to a stretching program. The animals had their muscles stretched for 30 s with 30 s of rest, with 10 repetitions, three and five times a week for 21 days. For morphological analysis, the sections were stained with hematoxylin-eosin and toluidine blue. For biochemical analysis, the tendons were treated with 4 M guanidine hydrochloride and analyzed in SDS-PAGE. The contents of total proteins and glycosaminoglycans were also measured. In the sections stained with toluidine blue, we could observe an increase of rounded cells, especially in the enthesis region. In the region next to the enthesis was a metachromatic region, which was more intensely stained in the stretched groups. In the tension regions, the cells appeared more aligned. Cellularity increased in both regions. The SDS-PAGE analysis showed a larger amount of collagen in the stretched groups and a polydispersed component of 65 kDa in all the groups. The amounts of proteins and glycosaminoglycans were also larger in the stretched tendons. The agarose-gel electrophoresis confirmed the presence of dermatan sulfate in the tension and compression regions, and of chondroitin sulfate only in the latter. Our results showed that the stretching stimulus changed the cellularity and the amount of the extracellular matrix compounds, confirming that tendons are dynamic structures with a capacity to detect alterations in their load.

Effects of passive stretching on the biochemical and biomechanical properties of calcaneal tendon of rats.

The role of physical activity in affecting the composition of extracellular matrix and mechanical properties of tendons has been well studied, but little is known about the role of passive stretching. The purpose of this study was to test the hypothesis that stimulation by passive stretching may change the composition and mechanical properties of tendons. Three-month-old Wistar rats were divided into three groups: the control, animals were not submitted to stretching procedures; groups that had their calcaneal tendons manually stretched three or five times a week, for 21 days. Afterward, the calcaneal tendons were removed and assayed for hydroxyproline content and biomechanical test. The hydroxyproline content in the stretched groups was higher, suggesting that more collagen was present in the tendons of these groups. These tendons also showed higher values of maximum stress and modulus of elasticity or Young's modulus. These results indicate that stretching leads to alterations in the synthesis of the extracellular matrix components and in the mechanical properties of tendons.