Effect of AAV9-synuclein treatment on the LC.

<p>Quantification of TH immunohistochemistry in the locus coeruleus using unbiased stereological techniques. Graph shows that 4 months following α-synuclein gene transfer there is a significant loss of TH positive cells in the LC (2-way ANOVA did not show a significant interaction, but did reveal main effects of diet and treatment, and bonferonni post-hoc revealed a difference between the α-synuclein control and diet treated groups, p<0.01). Treatment with spirulina was able to prevent the loss of TH positive cells in the locus coeruleus. Asterisk denotes significance (**p<0.01 vs control GFP; *p<0.05 vs control α-synuclein).</p

Effect of spirulina on NeuN immunoreactive cells in the SNpc.

<p>NeuN positive cells in the SNpc after 1 or 4 months of α-synuclein expression. At 1 month, there was a decrease in NeuN positive cells in the SN, mirroring the loss of TH positive cells in Fig. 1. This confirms cell loss rather than loss of TH expression. The effect was similar at 4 months of expression (B). There was neuroprotection in the groups that received a diet enhanced with spirulina at both intervals. Two-way ANOVA yielded a significant interaction of diet and vector treatment at both time points [1 month F = 6.931, df = 1; 4 months F = 8.899 df = 1]. (*p<0.05; **p<0.01) after Bonferonni's post-hoc.</p

Effects of spirulina on CX3CR1.

<p>Spirulina diet increased expression of CX3CR1; inset of Western blot in upper right. When the data are analyzed across groups there is a significant increase in expression of CX3CR1 in the spirulina treatment groups. Asterisk denotes significance (p<0.05; Bonferroni post-hoc). Two-way ANOVA shows a main effect of diet (F = 19.90; df = 1) and no interaction or main effect of vector treatment.</p

Gene transfer efficiency is not affected by spirulina.

<p>Quantification of GFP positive cells in the SN. Stereological estimates of the number of GFP positive cells in the SN one month after gene transfer. There was no significant effect of the spirulina diet on numbers of GFP transduced cells (Student’s two-talied t-test).</p

Effect of spirulina on TH immunoreactive cells in the SNpc.

<p>TH positive cells in the SNpc after 1 or 4 months of α-synuclein expression. Cells were counted using unbiased stereological counting techniques. (A) One month after α-synuclein gene transfer, there was a significant decrease in TH positive cells as compared to GFP control (N = 12–18/group). The spirulina diet group lesioned with α-synuclein (N = 12) had greater numbers of TH positive cells compared to the control diet group lesioned with α-synuclein (N = 18). There was a diet by vector group interaction in the two way ANOVA analysis [F = 5.569, p<0.01]. (B) Results at four months were similar. There was a similar loss of TH positive cells and protective effect of spirulina. Two-way ANOVA yielded a main effect of diet (F = 81.3), and a main effect of vector group (F = 45.5; p<0.01), although without a significant interaction. Bonferonni post-hoc tests comparing NIH 31 GFP (N = 10) vs NIH 31 synuclein (N = 8) and NIH31 synuclein vs spirulina synuclein (N = 8) groups were significant). Asterisk denotes significance (*P<0.05; **p<0.01).</p

Effect of MNC hUCB cell administration on lifespan of G93A mice.

<p>A) Kaplan-Meier survival curves for G93A mice receiving 10×10⁶, 25×10⁶, and 50×10⁶ MNC hUCB cells. Control groups were Media and CsA. Significant (four pointed star) increase in survival between cell transplanted and Media-injected or CsA-injected groups was determined in mice receiving 25×10⁶ cells (χ² = 4.134, p = 0.04). B) Some animals (33.3%) from the group receiving 25×10⁶ cells were alive at 20 wks of age, when only 21.4% of the group receiving 10×10⁶ cells and 7.7% of the group receiving 50×10⁶ cells survived. G93A mice administered with 25×10⁶ cells survived until 25 wks of age (22 wks of age–25%, 23 wks of age–16.7%, 25 wks of age–8.3%). Media-injected or CsA-injected mice survived no longer than 20 weeks of age.</p

Cytokine profile in the spleen of G93A mice administered with different MNC hUCB cell doses.

<p>The RNase protection assay was used to determine the mRNA expression of proinflammatory cytokines (IL-1 α, IL-1 β, TNF α, TNF β, and IL-2) and anti-inflammatory cytokine IL-10 in the spleen of G93A mice administered with different MNC hUCB cell doses. Control groups were Media, CsA, hTgn, and C57BL/6 mice. The mRNA expression presented as the optical density (OD) values obtained from each band normalizing against the OD obtained from the L32, a house-keeping gene, band. Lines indicated significant differences (p<0.001, p<0.01, and p<0.05) between mouse groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002494#s2" target="_blank">Results</a>).</p

Cytokine profile in the lumbar spinal cord, brainstem, and motor cortex of G93A mice administered with different MNC hUCB cell doses.

<p>The RNase protection assay was used to determine the mRNA expression of proinflammatory cytokines IL-1 α, IL-1 β, TNF α, and TNF β in the lumbar spinal cord (left column), brainstem (center column), and motor cortex (right column) of G93A mice administered with different MNC hUCB cell doses. Control groups were Media, CsA, hTgn, and C57BL/6 mice. The mRNA expression presented as the optical density (OD) values obtained from each band normalizing against the OD obtained from the L32, a house-keeping gene, band. Lines indicated significant differences (p<0.001, p<0.01, and p<0.05) between mouse groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002494#s2" target="_blank">Results</a>).</p

Immunochistochemical staining of MNC hUCB cells in the lumbar spinal cord of G93A mice administered with different cell doses.

<p>MNC hUCB cells were found in the lumbar spinal cord of mice receiving A), B) 10×10⁶; C), D) 25×10⁶; and E), F) 50×10⁶ cells by anti-human nuclei staining (green, asterisks). Merged images are with DAPI. Some a), b), c), d), e), f) MNC hUCB cells expressed Nestin (red, asterisks). Cells in images a, b, c, d, e, f are same in images A, B, C, D, E, F. Scale bar: A–e is 25 µm.</p

Human Th1/Th2 cytokines in plasma from G93A mice administered with different MNC hUCB cell doses.

<p>FAST Quant microspot assay for human Th1/Th2 Cytokines (IL-1 β, TNF α, INF γ, IL-2, IL-4, IL-5, IL-6, IL10, and IL-13) in plasma from G93A mice receiving three different cell doses was performed by Schleicher & Schuell BioScience, Inc. (Keene, NH, USA). Plasma samples from hUCB (n = 10) were used as controls. Quantitative results of cytokines were presented as pg/mL. The 50% of mice receiving 25×10⁶ cells showed evidence of human IL-4 and IL-13 (Th2). In 62.5% of mice with 50×10⁶ cell treatment, all human cytokines were detected except for IL-5, IL-5, and IL-10 (Th2). There were no human cytokines detected in mice treated with 10×10⁶ cell dose. The hUCB plasmas themselves, mostly contained Th2 cytokines (IL-4, IL-10, and IL-13).</p