A Combination of Schwann-Cell Grafts and Aerobic Exercise Enhances Sciatic Nerve Regeneration

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.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.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.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.These data provide evidence that this combination of therapeutic strategies can significantly improve functional and morphological recovery after sciatic injury

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

Quantification of neurons nucleoli in spinal cord and in dorsal root ganglion (D).

<p>Cross sections through the spinal cord (A–D) and longitudinal sections through DRG (F–I), 8 weeks after sciatic nerve transection. Arrows show examples of quantified nucleoli. (E) and (J) represent quantification of motor neurons in the ventral horn spinal cord and sensory neurons in DRG. Values shown are mean ± SE; *<i>P<0.05</i>. Scale bars: 50 µm.</p

Scanning electron microscopy of polycaprolactone tubes.

<p>(A and A′) Appearance of the empty tube before being implanted (A), and higher magnification of the tube wall showing its normal aspect (A′); (B and B′) tube containing the growing nerve in its interior (B) and the tube wall starting to disintegrate three weeks after the implant (B′); (C and C′) regenerated nerve within the tube (C′) and tube wall in process of disintegration (C′) eight weeks after implantation. Arrows delimit the thickness of the tube wall, which is clearly reduced over time. Bar: 200 µm (A, B and C) and 50 µm (A′, B′ and C′).</p

Combined therapies improve nerve regeneration.

<p>(A–D) Semi-thin cross sections of regenerating sciatic nerves. (A) DMEM group with clusters of regenerating nerve fibers (arrowhead), myelinated fibers (large arrow) and blood vessels (thin arrow). (B, C and D) TMT, SC and TMT + SC-treated groups display nerves with many clusters of regenerating fibers (arrowheads) with myelinated nerve fibers (thick arrows) and blood vessels (thin arrows). Scale bar  = 20 µm. (E–H) Transmission electron micrographs of regenerating nerves. (E) DMEM group; cross section showing small and poorly developed regenerating clusters (arrowhead) composed of thin, dispersed myelinated nerve fibers (thick arrow) and non-myelinated nerve fibers (thin arrow). (F, G and H) TMT, SC and TMT + SC-treated groups showing regenerating clusters consisting of myelinated (thick arrows) and non-myelinated nerve fibers (thin arrows) surrounded by processes of perineurium-like cells (arrowheads). Schwann-cell nuclei are also observed (asterisks). Scale bar  = 2 µm.</p

Functional analysis of global mobility test (GMT).

<p>(A and B) DMEM group; (C and D) TMT group; (E and F) SC group; (G and H) TMT + SC group; at different times after surgery (14 and 56 days). In general, treated groups showed improved exploratory capacity through the open field. (I) GMT analysis indicated that TMT and TMT + SC-treated animals were able to move faster compared to the DMEM and SC-treated animals. Values represent mean ± SEM, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Functional recovery is improved with TMT + SC combined therapy.

<p>(A) Measurements of print length (PL) and toe spread (TS), exemplifying the values used to measure the footprint. (B) Line graph showing the variation of the sciatic functional index (SFI) over time. (C) Bar graph (normalized to positive values), showing statistically significant differences between the treated groups and DMEM (represented by *); between the TMT and TMT + SC groups (represented by #), and between the TMT + SC and SC groups (represented by +). The TMT + SC group had a better functional outcome, indicated by the significant difference at 7 days after injury.</p

Quantitative analyses of the number of myelinated nerve fibers, myelin area, axon area and fiber area.

<p>(A–D; *<i>P</i><0.05) (E) Quantitative analysis of the number of blood vessels. (F) G-ratio analyses stratified by ranges. Values represent mean ± SEM, *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p

Quantification of relative immunoreactive area stained with BDNF, NGF, NT3 and NT4 in the sciatic nerve, dorsal root ganglion and spinal cord (A–L).

<p>Analysis of BDNF, NGF and NT4 expression on sciatic nerve showed significant values when the TMT + SC group was compared to the DMEM group (A–D). The DRG quantification on the TMT + SC group presented significantly higher levels of all analyzed trophic factors in relation to the DMEM group (E–H). Trophic factor staining in spinal cord presented a significant difference in the NT4 fluorescence intensity between TMT + SC and DMEM groups (I–L). Values represent mean ± SEM, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Motoneurons regenerate and form neuromuscular junctions.

<p>Longitudinal sections of gastrocnemius muscle showing end plates identified with α-bungarotoxin binding to acetylcholine receptors (red) and nerve terminal stained with neurofilament-200 (green). (A and B) Normal animal used as positive control; most end plates exhibited a “pretzel-like” shape with precise apposition of motor nerve terminals to the motor endplates, indicating that the endplate is perfectly innervated. (C, D and E) DMEM, TMT and SC groups showed neuromuscular junctions with disorganized and enlarged morphology, and exhibited poor and fragmented NF-200 staining. (F) The TMT + SC group displayed more “pretzel-like” junctions, similar to normal animal. Bar: (A) 50 µm (B, C, D, E and F) 25 µm.</p