Spirulina Promotes Stem Cell Genesis and Protects against LPS Induced Declines in Neural Stem Cell Proliferation

Adult stem cells are present in many tissues including, skin, muscle, adipose, bone marrow, and in the brain. Neuroinflammation has been shown to be a potent negative regulator of stem cell and progenitor cell proliferation in the neurogenic regions of the brain. Recently we demonstrated that decreasing a key neuroinflammatory cytokine IL-1β in the hippocampus of aged rats reversed the age-related cognitive decline and increased neurogenesis in the age rats. We also have found that nutraceuticals have the potential to reduce neuroinflammation, and decrease oxidative stress. The objectives of this study were to determine if spirulina could protect the proliferative potential of hippocampal neural progenitor cells from an acute systemic inflammatory insult of lipopolysaccharide (LPS). To this end, young rats were fed for 30 days a control diet or a diet supplemented with 0.1% spirulina. On day 28 the rats were given a single i.p. injection of LPS (1 mg/kg). The following day the rats were injected with BrdU (50 mg/kg b.i.d. i.p.) and were sacrificed 24 hours after the first injection of BrdU. Quantification of the BrdU positive cells in the subgranular zone of the dentate gyrus demonstrated a decrease in proliferation of the stem/progenitor cells in the hippocampus as a result of the LPS insult. Furthermore, the diet supplemented with spirulina was able to negate the LPS induced decrease in stem/progenitor cell proliferation. In a second set of studies we examined the effects of spirulina either alone or in combination with a proprietary formulation (NT-020) of blueberry, green tea, vitamin D3 and carnosine on the function of bone marrow and CD34+ cells in vitro. Spirulina had small effects on its own and more than additive effects in combination with NT-020 to promote mitochondrial respiration and/or proliferation of these cells in culture. When examined on neural stem cells in culture spirulina increased proliferation at baseline and protected against the negative influence of TNFα to reduce neural stem cell proliferation. These results support the hypothesis that a diet enriched with spirulina and other nutraceuticals may help protect the stem/progenitor cells from insults

A spirulina-enhanced diet provides neuroprotection in an α-synuclein model of Parkinson's disease.

Inflammation in the brain plays a major role in neurodegenerative diseases. In particular, microglial cell activation is believed to be associated with the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). An increase in microglia activation has been shown in the substantia nigra pars compacta (SNpc) of PD models when there has been a decrease in tyrosine hydroxylase (TH) positive cells. This may be a sign of neurotoxicity due to prolonged activation of microglia in both early and late stages of disease progression. Natural products, such as spirulina, derived from blue green algae, are believed to help reverse this effect due to its anti-inflammatory/anti-oxidant properties. An adeno-associated virus vector (AAV9) for α-synuclein was injected in the substantia nigra of rats to model Parkinson's disease and to study the effects of spirulina on the inflammatory response. One month prior to surgeries, rats were fed either a diet enhanced with spirulina or a control diet. Immunohistochemistry was analyzed with unbiased stereological methods to quantify lesion size and microglial activation. As hypothesized, spirulina was neuroprotective in this α-synuclein model of PD as more TH+ and NeuN+ cells were observed; spirulina concomitantly decreased the numbers of activated microglial cells as determined by MHCII expression. This decrease in microglia activation may have been due, in part, to the effect of spirulina to increase expression of the fractalkine receptor (CX3CR1) on microglia. With this study we hypothesize that α-synuclein neurotoxicity is mediated, at least in part, via an interaction with microglia. We observed a decrease in activated microglia in the rats that received a spirulina- enhanced diet concomitant to neuroprotection. The increase in CX3CR1 in the groups that received spirulina, suggests a potential mechanism of action

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

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

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

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