Characteristics of motor neuron survival in the spinal cord of G93A mice.

<p>Motor neuron counts were performed in the cervical (<b>A</b>) and lumbar (<b>B</b>) ventral horns of G93A mice at 17 weeks of age and at end-stage of disease. Mice receiving 2.5×10⁶ cells symptomatically (<i>Gr 1</i>) or 1×10⁶ cells pre-symptomatically (<i>Gr 3</i>) had significantly higher motor neuron densities than the Media group (<i>Gr 4</i>) at 17 weeks of age or at end-stage of disease. In both cervical and lumbar spinal cords, motor neuron densities between <i>Gr 2</i> (1×10⁶ cells, symptomatic) and Media-injected (<i>Gr 4</i>) mice showed no significant differences (p>0.05). *p<0.05, **p<0.01, ***p<0.001. (<b>C</b>) Immunohistochemical staining of motor neurons in the lumbar spinal cord of G93A mice at 17 weeks of age. Motor neuron staining for anti-choline acetyltransferase (anti-ChAT) antibody showed healthy motor neurons in controls (<b>a</b>) although only a few neurons survived in the Media-treated animals (<b>b</b>). Cell-treated mice with (<b>c</b>) 2.5×10⁶ cells symptomatically (<i>Gr 1</i>) and (<b>e</b>) 1×10⁶ cells pre-symptomatically (<i>Gr 3</i>) demonstrated higher motor neuron survival than (<b>d</b>) mice receiving 1×10⁶ cells symptomatically (<i>Gr 2</i>). Scale bar: a–e is 50 µm.</p

Characteristics of microglial cells in the spinal cords of G93A mice.

<p>Microglial densities were measured in the cervical (<b>A</b>) and lumbar (<b>B</b>) ventral horns of G93A mice at 17 weeks of age and at end-stage of disease. Microglial densities were significantly (p<0.001) higher in Media-injected mice (<i>Gr 4</i>) compared to control mice (<i>Gr 5</i>) of the same ages. MNC hUCB cell administrations significantly (p<0.001) decreased the number of microglia in the spinal cord of G93A mice compared to Media mice. No significant differences were detected between the cell-treated groups. *p<0.05, **p<0.01, ***p<0.001. (<b>C</b>) Immunohistochemical staining of microglia in the lumbar spinal cord at 17 weeks of age. Microglial cells stained for anti-Iba-1 antibody were sparse in controls (<b>a</b>) and microgliosis was noted in Media-treated animals (<b>b</b>). MNC hUCB cells decreased microglial density in mice from <i>Gr 1</i> (<b>c</b>), <i>Gr 2</i> (<b>d</b>), and <i>Gr 3</i> (<b>e</b>). Morphological analysis of microglial cells determined numerous activated cells with large cell bodies and short processes in Media-injected mice, whereas ramified microglia were mostly observed in cell-treated animals, particularly in <i>Gr 1</i> and <i>Gr 3</i> mice and controls (inserts in a–e). Scale bar: a–e is 200 µm; in a–e inserts is 25 µm.</p

Distribution of MNC hUCB cells in the spinal cord of G93A mice.

<p>Administered MNC hUCB cells were identified immunohistochemically by a human-specific marker (HuNu) in the spinal cord of cell-treated mice at 17 weeks of age, 4 weeks (symptomatic) or 8 weeks (pre-symptomatic) post-transplant. In the total area of cervical (<b>A</b>) and lumbar (<b>B</b>) cervical spinal cord, HuNu positive MNC hUCB cells were found irrespective (p>0.05) of injected cell doses or time beginning treatment. In all cell-treated mice, more than 50% of the observed cells were in ventral horn gray matter. (<b>C</b>) Immunohistochemical staining of MNC hUCB cells in the lumbar spinal cord. MNC hUCB cells positive for HuNu (green, arrow) were detected in the lumbar spinal cord of mice receiving 2.5×10⁶ (<b>a</b>) or 1×10⁶ (<b>b</b>) cells symptomatically or 1×10⁶ cells pre-symptomatically (<b>c</b>). Cells were frequently observed inside the capillary lumen, but also in the spinal cord parenchyma. (<b>a′</b>), (<b>b′</b>), and (<b>c′</b>) are merged images with DAPI. Scale bar: a–c′ is 50 µm.</p

Evaluations of disease progression in G93A mice through Kaplan-Meier analysis.

<p>(<b>A</b>) Time elapsed until animals lost 15% of their maximum body weight. Mice receiving 1×10⁶ cells pre-symptomatically (<i>Gr 3</i>) significantly () maintained body weight vs. Media (<i>Gr 4</i>) mice. A similar trend was observed in mice treated with 2.5×10⁶ cells (<i>Gr 1</i>) beginning at symptomatic disease stage. (<b>B</b>) Time elapsed until hindlimb extension scores deteriorated by 70% of the initial score. The <i>Gr 1</i> and <i>Gr 3</i> mice significantly () delayed decline of hindlimb extension compared to <i>Gr 4</i> mice. A significant difference was also detected between <i>Gr 3</i> and <i>Gr 2</i> mice receiving 1×10⁶ cells at pre-symptomatic or symptomatic stage of disease, respectively. (<b>C</b>) Time elapsed until muscle strength decreased by 70% from the maximum value. Mice from <i>Gr 3</i> significantly () delayed muscle strength losses vs. <i>Gr 4</i>. <i>Gr 1</i> mice tended to maintain muscle strength post-transplant. (<b>D</b>) Time elapsed until rotarod latency decreased by 70% of the maximum value. Only mice from <i>Gr 3</i> performed better on the rotarod than other cell-treated mice and tended towards significance () taking more time to decrease latency by over 70% of the maximum value compared to <i>Gr 4</i>.</p

Effect of multiple MNC hUCB cell administrations on lifespan of G93A mice.

<p>(<b>A</b>) Kaplan-Meier survival curves for G93A mice receiving 2.5×10⁶ (<i>Gr 1</i>) or 1×10⁶ (<i>Gr 2</i>) cells at symptomatic disease stage and 1×10⁶ (<i>Gr 3</i>) cells pre-symptomatically. Control group was Media-injected mice (<i>Gr 4</i>). Significant () increases in survival were determined in mice receiving 1×10⁶ cells at pre-symptomatic stage (p = 0.0015) and 2.5×10⁶ cells at symptomatic stage (p = 0.0022) vs. the Media-injected group. Survival of the mouse group receiving 1×10⁶ cells pre-symptomatically tended towards significance compared to survival of mice receiving same cell dose at symptomatic stage (p = 0.0595). (<b>B</b>) Percentages of surviving mice within age ranges. Media-injected animals survived no longer than 19.5 weeks, whereas 30% of mice receiving 2.5×10⁶ (<i>Gr 1</i>, symptomatic) or 1×10⁶ cells (<i>Gr 3</i>, pre-symptomatic) and 14.3% mice administered with 1×10⁶ cells (<i>Gr 2</i>) at symptomatic stage survived more than 140 days and 10% of mice from <i>Gr 3</i> (1×10⁶ cells, pre-symptomatic) were alive for more than 150 days.</p

Distribution of MNC hUCB cells in the lung, liver, kidney and spleen of G93A mice.

<p>MNC hUCB cells immunohistochemically positive for HuNu (green, arrows) were detected in the lung, liver, kidney, and spleen of mice receiving 2.5×10⁶ (<b>a, d, g, j</b>) or 1×10⁶ (<b>b, e, h, k</b>) cells symptomatically or 1×10⁶ cells pre-symptomatically (<b>c, f, i, l</b>). In the liver, lung and kidney, few cells were identified. In the spleen, a high density of HuNu cells was determined in all cell-treated mice (<b>j–l</b>). Scale bar: a–i is 50 µm; j–l is 200 µm.</p

BrdU found in microglia only in the control fed mice following LPS.

<p>Low magnification confocal photomicrographs were used to determine if the BrdU was labeling non-neural cells. (<b>A–C</b>) In the control animals without LPS the majority of the BrdU cells were not found to be microglia. (<b>D–E</b>) In the control diet fed rats treated with LPS note that fewer BrdU cells were found and that many of the BrdU labeled cells were microglia. White arrow shown in high magnification in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010496#pone-0010496-g003" target="_blank">Fig. 3D–F</a> Yellow arrow shown in high magnification in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010496#pone-0010496-g003" target="_blank">Fig. 3G–I</a>. (<b>G–I</b>) In the spirulina fed rats the BrdU cells were not found to be microglia.</p

Spirulina rats have a decreased astrocyte response to LPS.

<p>(<b>A</b>) LPS caused a significant increase in the percentage of the dentate gyrus which was GFAP positive. The spirulina fed rats had a reduced GFAP response LPS compare to the control diet fed rats with LPS. *** p = 0.0002 (NIH-31 vs. NIH-31+LPS). ††† P = 0.0003 (NIH-31+LPS vs. spirulina+LPS). (<b>B</b>) There was no significant difference in the total number of GFAP⁺ cells. (<b>C</b>) Representative photomicrograph of the GFAP immunohistochemical staining in the hippocampus. The white arrow points to the area shown in (<b>D</b>) at 20× magnification.</p

Orthogonal projections of BrdU IBA1 co-staining.

<p>(<b>A–C</b>) shows a representative cluster of BrdU⁺ cells in the SGZ confirming the cells are not microglia by the lack of colocalization with IBA-1. (<b>D–E</b>) shows a cluster of BrdU⁺ cells in the LPS treated rats that are not microglia. (<b>G–I</b>) shows a BrdU⁺ that does double label with the microglia marker IBA-1 as noted by the yellow color in the merged panel. (<b>J–K</b>) shows BrdU and IBA-1 staining in the Spirulina fed rats. Note, in this and all confocal images the side panels for each box are 90° rotations of the Z-stacks for confirmation of double labeling.</p

Spirulina increases proliferation of human hematopoetic stem cells <i>in vitro</i>.

<p>(<b>A</b>) The effects of spirulina and NT-020 on the proliferation of human bone marrow cells was examined. Cells were plated in 96 well plates and spirulina at varying concentrations was added to the culture media. In a separate study shown in the same graph the highest dose of spirulina tested alone (125 ng/ml) was added to NT-020. After 72 hours in culture viability was tested using the MTT assay. Data is expressed at % over control, which are cells grown in media only. When spirulina is combined with NT-020 the effect is significantly higher than NT-020 alone was more than additive. (<b>B</b>) The effects of spirulina and NT-020 on the proliferation of human CD34⁺ cells was examined. Cells were plated in 96 well plates and spirulina at varying doses was added to the culture media, in a separate study shown in the same graph the highest dose of spirulina tested alone (125 ng/ml) was added to NT-020. After 72 hours in culture viability was tested using the MTT assay. Data is expressed at % over control, which is cells grown in media only. When spirulina is combined with NT-020 the effect is significantly higher than NT-020 alone (p<0.05 students 2-tailed t-test) and the effect appears to be additive. ** p<0.001 ***p<0.0001 (compare to control). P<0.0001 (NT-020 vs. spirulina+NT-020).</p