Estimated mean and 95% confidence interval of each group for spontaneous motor activity analysis.

<p>Estimated mean and 95% confidence interval of each group for spontaneous motor activity analysis.</p

Effect time and group for symmetric score by cylinder test.

<p>Effect time and group for symmetric score by cylinder test.</p

Blood perfusion image analysis of ischemic lesion area and TTC staining image (Experiment 1).

<p>A: Animal´s image during baseline perfusion analysis. B, E, H, and K: The blood perfusion values at basal and 30 min after craniectomy in the sham group (<i>n</i> = 5) and 30 min after TCI at 200°C, 300°C, and 400°C, respectively, in ischemic groups (<i>n</i> = 5 in each group), combined with coronal TTC-staining slices of animals 120 min after TCI in each condition (as seen in the upper left margin); C, F, I, and L: The blood perfusion basal images in each experimental condition; D: The blood perfusion image 30 min after craniectomy in sham condition; G, J, and M: The blood perfusion image 30 min after TCI at 200°C, 300°C, and 400°C, respectively; and N: Decrease in perfusion rate in each experimental condition, between the basal time (after craniectomy) and 30 min after TCI and the data were represented by the boxes that show the values of the 25th and 75th percentiles, the lines across the boxes represent the medians, and the whiskers extend to the highest and lowest values, using different shades of gray for each group.</p

Motor behavior assessment using Actimeter and cylinder tests at basal and after 7 days of thermocoagulation induction (Experiment 2).

<p>In Actimeter test (A-F), A: Slow movement (horizontal activity), B: Fast movement (horizontal activity), C: Slow stereotyped, D: Fast stereotyped, E: Slow rearing movement (vertical activity), and F: Fast rearing movement (vertical activity). The cylinder test (G-H), G: Bilateral forelimb analyses-on the left, an animal with bilateral symmetry and on the right, an animal with asymmetry contralateral with the ischemic lesion, H: Symmetry score at basal time and seven days after induction. Both tests were performed in the five experimental groups (n = 5 per group): control, sham, ischemic with 200°C, ischemic with 300°C, and ischemic with 400°C, which were represented by the boxes that show the values of the 25th and 75th percentiles, the lines across the boxes represent the medians, and the whiskers extend to the highest and lowest values, using different shades of gray for each group. *: p<0.001 in comparison with base time, #: p<0.001 in comparison with control group, and §: p<0.001 in comparison with the sham group.</p

Ischemic lesion induction by thermocoagulation (Experiments 1 and 2).

<p>A-I: A hot probe with digital control of temperature, A-II: Fiber optic thermometer systems, B–D: Thermocoagulation induction (TCI) at 200°C, 300°C, and 400°C (red arrow) and thermal dissipation analysis in the brain tissue by using fiber optic at each temperature condition (yellow arrow) (<i>n</i> = 5 per group), E–G: Thermal dissipation analysis on the tissue brain through infrared camera at each temperature of induction, H: color of brain tissue before TCI (light red), and I: color brain tissue after TCI (dark red).</p

Estimated mean for symmetric score by cylinder test.

<p>Estimated mean for symmetric score by cylinder test.</p

Additional file 1: Figure S1. of Intraspinal bone-marrow cell therapy at pre- and symptomatic phases in a mouse model of amyotrophic lateral sclerosis

Quantification of neurons in the anterior horn of the spinal cord. The number of interneurons (NeuN-positive cells with cross-sectional area ≤250 μm2) was analyzed in week 12 in the presymptomatic injected animals a and in week 15 in the symptomatic injected animals b. There was no difference in the number of interneurons in the SOD1G93A mice compared with the wild-type animals. The number of motor neurons was analyzed in the end stage of the disease in the presymptomatic injected animals c and in the symptomatic injected animals d. Both saline-injected and BMMC-injected mice showed a decrease in the number of motor neurons compared with the wild-type mice at the time point analyzed. ***p <0.001. (TIF 745 kb

Distribution of Mesenchymal Stem Cells and Effects on Neuronal Survival and Axon Regeneration after Optic Nerve Crush and Cell Therapy

<div><p>Bone marrow-derived cells have been used in different animal models of neurological diseases. We investigated the therapeutic potential of mesenchymal stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult (3–5 months old) Lister Hooded rats underwent unilateral optic nerve crush followed by injection of MSC or the vehicle into the vitreous body. Before they were injected, MSC were labeled with a fluorescent dye or with superparamagnetic iron oxide nanoparticles, which allowed us to track the cells <i>in vivo</i> by magnetic resonance imaging. Sixteen and 28 days after injury, the survival of retinal ganglion cells was evaluated by assessing the number of Tuj1- or Brn3a-positive cells in flat-mounted retinas, and optic nerve regeneration was investigated after anterograde labeling of the optic axons with cholera toxin B conjugated to Alexa 488. Transplanted MSC remained in the vitreous body and were found in the eye for several weeks. Cell therapy significantly increased the number of Tuj1- and Brn3a-positive cells in the retina and the number of axons distal to the crush site at 16 and 28 days after optic nerve crush, although the RGC number decreased over time. MSC therapy was associated with an increase in the FGF-2 expression in the retinal ganglion cells layer, suggesting a beneficial outcome mediated by trophic factors. Interleukin-1β expression was also increased by MSC transplantation. In summary, MSC protected RGC and stimulated axon regeneration after optic nerve crush. The long period when the transplanted cells remained in the eye may account for the effect observed. However, further studies are needed to overcome eventually undesirable consequences of MSC transplantation and to potentiate the beneficial ones in order to sustain the neuroprotective effect overtime.</p></div

MSC transplantation increases FGF-2 and IL-1β expression in the retinal ganglion cell layer.

<p>(A-H) Confocal images of FGF-2 and IL-1β expression in retinas from vehicle injected (A-D) and MSC treated (E-H) groups. TOPRO (C, G) was used for nuclei staining. Images are representative of 3 animals per experimental condition. Scale Bar: 20 µm. (I, J) Graphs show the average mean gray value of Z stack images normalized by the vehicle injected group. *p<0.05, unpaired t-test. RGCL: retinal ganglion cell layer. Scale bar: 50 µm.</p

MSC transplantation increased RGC axon regeneration 16 and 28 days after optic nerve crush.

<p>A-D: Photomontage of confocal images of optic nerve sections to illustrate axonal outgrowth. CTB-488 was injected into the vitreous body 2 days prior to euthanasia to label the axons. Sixteen days after nerve crush (A,B,E) only a few axons had crossed the lesion site (asterisk) in the vehicle-injected animals (A) and this number was larger in the MSC-injected animals (B). Twenty-eight days after optic nerve injury (C,D,F), the MSC-injected group had an even larger number of axons regenerating beyond the crush site, compared to the vehicle-injected group. E, F: Quantification of CTB-488⁺ axons per nerve at different distances from the crush site (from 0.25 to 2.0 mm). Results are displayed as mean ± SEM. *P<0.05; **P<0.01. Scale bar: 200 µm.</p