Mesenchymal Stem Cell Graft Improves Recovery after Spinal Cord Injury in Adult Rats through Neurotrophic and Pro-Angiogenic Actions

Numerous strategies have been managed to improve functional recovery after spinal cord injury (SCI) but an optimal strategy doesn't exist yet. Actually, it is the complexity of the injured spinal cord pathophysiology that begets the multifactorial approaches assessed to favour tissue protection, axonal regrowth and functional recovery. In this context, it appears that mesenchymal stem cells (MSCs) could take an interesting part. The aim of this study is to graft MSCs after a spinal cord compression injury in adult rat to assess their effect on functional recovery and to highlight their mechanisms of action. We found that in intravenously grafted animals, MSCs induce, as early as 1 week after the graft, an improvement of their open field and grid navigation scores compared to control animals. At the histological analysis of their dissected spinal cord, no MSCs were found within the host despite their BrdU labelling performed before the graft, whatever the delay observed: 7, 14 or 21 days. However, a cytokine array performed on spinal cord extracts 3 days after MSC graft reveals a significant increase of NGF expression in the injured tissue. Also, a significant tissue sparing effect of MSC graft was observed. Finally, we also show that MSCs promote vascularisation, as the density of blood vessels within the lesioned area was higher in grafted rats. In conclusion, we bring here some new evidences that MSCs most likely act throughout their secretions and not via their own integration/differentiation within the host tissue

Neuroprotection.

<p>MSC transplants favor tissue sparing after SCI. Luxol fast blue/hematoxylin staining on cross sections of MSC-grafted (<b>A</b>) and control injured only (<b>B</b>) rats, 21 days after transplantation. The quantification of spared tissue (<b>C</b>), as assessed by the mean ratio of injured area on total area of the sections, reveals a significant decrease of the lesion extension in MSC treated rats compared to control ones. * p<0.05.</p

Mesenchymal stem cell characterization.

<p>(<b>A</b>): Phase contrast microphotography of rat MSCs in culture. Cells exhibit typical elongated, fibroblast-like morphology (arrowheads) or large, flattened shape (asterisks). (<b>B</b>) CD90 and (<b>C</b>) CD271 positive immunofluorescent labelings of P4 MSCs in culture, while CD45 <b>(D)</b> and CD11b (<b>E</b>) immunostainings are negative. (<b>F</b>) Adipogenic differentiation of P4-MSCs revealed with Oil Red O. (<b>G</b>) Alizarin Red staining of P4 MSCs induced to osteocytes reveals mineral deposition. (<b>H</b>) Immunofluorescent visualization of nestin induction in P12-MSCs deprived of serum. Nuclei are labeled with the Vectashield-DAPI mounting medium.</p

Behavioral analysis.

<p>(<b>A</b>) Locomotor scores as assessed by the BBB rating scale. MSC grafted rats reach significantly higher scores compared to both control groups. Only MSC transplanted rats reach the weight-supporting step level (score of 9). Injured + MSC vs injured only p = 0.0029; injured + MSC vs injured + vehicle p<0.0001. (<b>B</b>) Grid test scores assessing deficits in descending fine motor control. MSC grafted rats reach higher scores, significantly different from control rats. Injured + MSC vs injured only p<0.0001; injured + MSC vs injured + vehicle p<0.0001.</p

Cytokine array and Elisa results.

<p>(<b>A</b>) Cytokine arrays. Spinal cord extracts from injured vehicle-treated (n = 4) and MSC-treated (n = 5) rats. ß-NGF is significantly increased within the lesioned site 3 days after MSC injection compared to controls. * p<0.05 (mean±S.E.). (<b>B</b>) Histogram showing the amounts, in pg/ml, of the neurotrophins NGF and BDNF as quantified by Elisa, within 2 distinct P12 MSC-conditioned media (M1 and M2). (<b>C</b>) NGF and (<b>D</b>) BDNF fluorescent immunocytochemistry on P12 MSCs <i>in vitro</i>. Scale bar : 50 µm.</p

Blood vessel quantification.

<p>(<b>A</b>–<b>B</b>) RECA-1 immunohistological staining on longitudinal sections of MSC-treated (<b>A</b>) and control vehicle-treated (<b>B</b>) spinal cords. Scale bar: 500 µm. (<b>C</b>) Blood vessel quantification within the lesioned site reveals a significant increase in MSC treated rats compared to control-vehicle treated ones. *p<0.05.</p

Axonal regrowth.

<p>(<b>A</b>–<b>B</b>) GAP43 immunoreactivity on transversal spinal cord sections, 28 days after SCI, in injured + MSC (A) and injured-only (B) groups. Sections were taken at the lesion site. (<b>C</b>) Image analysis doesn't show a significant difference (Student's t-Test, p = 0,23) in the percentage of total lesioned area immunoreactive for GAP43 between treated (2,96 ± 0,35 %) and control (2,25 ± 0,44 %) groups</p

BrdU immunodetection.

<p>(<b>A</b>) BrdU (FITC)-DAPI immunostaining on MSCs cultured with 1.10⁻⁶M BrdU for 72 h, demonstrating that cells were all labeled before being transplanted. (<b>B–C–D</b>) BrdU (Rhodamine) maintenance in MSCs 3 days (<b>B</b>), 7 days <b>(C)</b> and 21 days (<b>D</b>) after the removal of BrdU from the culture medium. (<b>E</b>) BrdU immunodetection (FITC) on a longitudinal spinal cord tissue section from a rat that received 3 ip BrdU injections after spinal cord injury. Scale bar: 50 µm (A), 100 µm (B, C, D) and 200 µm (E).</p

MSC viability and CD profile.

<p>Nestin (<b>A</b>) and p75NGFr (<b>B</b>) immunofluorescent stainings on MSCs: The last drop of MSC suspension from the injection needle was placed back into culture to check their viability at the time of injection. (<b>C</b>–<b>F</b>): CD profile of P12 MSC grafted cells, showing their immunoreactivity for CD90 (C) and CD271 (D), and the absence of CD45 (E) and CD11b (F) expression. Scale bar: 50 µm (A, B), 100 µm (C–F).</p