Abstract Top

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.

Introduction Top

Neurogenesis is a life-long occurrence that is limited to specific sites within the brain; namely, the subventricular zone (SVZ), and the subgranular zone (SGZ) of the hippocampus. The de novo production of new neurons into the hippocampus, has been shown to be important for some forms of learning [1]. While numerous studies have shown that neurogenesis is physiologically relevant for cognitive function, the relationship is complex (For review see: [2], [3]). Nonetheless, neurogenesis is clearly linked to plasticity and repair mechanisms [4] and alterations in neurogenesis have been also been attributed to some affective disorders [5].

Two seminal studies, published simultaneously, a number of years ago showed that inflammation tightly regulates neurogenesis in the SGZ [6], [7]. Ekdahl et al. (03) used LPS that they delivered into the cortex continuously by an osmotic mini pump. In the young adult rat, LPS-induced inflammation resulted in an 85% reduction in the number of new neurons born during the inflammatory insult [6]. Monje et al. (03) also found that LPS given systemically caused an increase in microglia activation and a decrease in neurogenesis, which could be prevented by the nonsteroidal anti-inflammatory drug (NSAID) indomethacin [7].

Cytokines do appear capable of regulating several phases of the neurogenesis process. At low concentrations the proinflammatory cytokine TNF-α induces proliferation of neural progenitor cells (NPC), but at higher concentrations TNF-α results in program cell death [8]. TNF-α induced program cell death in the NPC is dependent on TNF receptor 1 (TNF-RI) [9] which is constitutively expressed by NPC in culture [10]. IL-1β can also directly suppress neurogenesis by blocking the production of cyclic dependent kinesis [10], [11]. Inflammation also alters the way the new neurons integrate into the existing neuronal circuit [12].

We have previously demonstrated that spirulina can act as an anti-inflammatory, and reverse the age-related elevation of TNF-α [13]. Activation of TNF-RI by TNF-α negatively regulates the proliferation of NPC [10]. Therefore we hypothesized that spirulina could protect against an acute systemic inflammatory insult induced by LPS the effects would occur during the proliferative phase of neurogenesis. To test this hypothesis, young adult Fisher 344 rats were fed a control diet, or a diet supplemented with 0.1% spirulina for 28 days before administration of LPS. As predicted, LPS decreased proliferation of the hippocampal NPCs. Spirulina was able to prevent the LPS induced decrease in NPC proliferation. The LPS insult did not appear to have pronounced affects on microglial activation but did produce a measurable increase in astrogliosis. Pretreatment with spirulina also blocked the LPS induced astrogliosis. The in vivo results suggest that spirulina protects by a non-stem cell autonomous mechanisms.

Previous studies have shown that nutraceuticals can have effects on adult stem cells. A nutraceutical combination of blueberry extract, green tea extract, carnosine, and vitamin D3 (a proprietary formulation known as NT-020) has been shown to promote migration of brain stem cells from the stem cell niche to the site of injury in an animal model of stroke [4]. NT-020 was shown to stimulate the proliferation of human stem cells derived from bone marrow; bone marrow derived CD34+ and progenitor cells from peripheral blood (CD133+) in vitro [14]. Cultured bone marrow cells removed from mice given NT-020 orally for 2 weeks exhibited a dose-related reduction of oxidative stress-induced cell death. This demonstrates that the action of this nutraceutical on stem cells is not dependent on the presence of the formulation as the effect was observed when the cells were cultured in the absence of NT-020 for 3 days.

To investigate if spirulina could promote proliferation of cells other than neural stem cells investigated the effects of spirulina both alone and in combination with NT-020 to promote the proliferation of bone marrow and CD34+ stem cells in culture as measured with MTT. We found that spirulina increased proliferation/mitochondrial respiration of CD34+ stem cells in culture. The results of this study demonstrate that spirulina can negate the negative effects of inflammation on neurogenesis, indirectly by decreasing astrogliosis and by potentially acting directly on the stem cells and promote proliferation of bone marrow and CD34+ human stem cells in culture.

Results Top

Spirulina protects against LPS induced decrease in hippocampal progenitor cell proliferation

Before induction of the LPS inflammatory insult, young adult male Fisher 344 rats were fed either a standard NIH-31 diet or a spirulina-enriched diet (NIH-31 +0.1% spirulina). After 28 days on the diets the rats were injected with LPS (1 mg/kg) into the peritoneal cavity to induce an acute inflammatory insult. The following day, day 29, the rats were injected twice (8 hours interval) with BrdU (50 mg/kg) to label those cells that were proliferating while BrdU was available. The rats were then euthanized on the following day to determine if spirulina could protect against the anti-neurogenic effects of LPS (see Fig. 1A for timeline). Quantification of the BrdU labeled cells, in the SGZ of the hippocampus, was performed using the optical fractionator method of design based stereology [15]. A significant effect was found (F(2,19) = 5.913, p+ cells in the SGZ in the rats euthanized two days after the inflammatory insult of LPS (Fig. 1A). As we predicted, LPS did significantly decrease the number of BrdU+ cells in the rats fed the control diet (t(10) = 3.589; p = 0.0049). The cytokine storm induced by LPS peaks 2 hours after intraperitoneal injection of LPS [16], we found that even one full day after the peak of the cytokine storm, proliferation of the NPC was reduced by nearly 40% in rats fed the control diet. A diet enriched with spirulina was able to completely prevent the LPS induced decrease in NPC proliferation. In rats fed a spirulina enriched diet prior to being injected with LPS, proliferation of NPC was not significantly different then the control fed rats that were not injected with LPS. Moreover, in comparison to the control fed rats that were injected with LPS, the spirulina fed rats that were also injected with LPS had significantly more BrdU labeled cells in the SGZ (t(12) = 3.113; p = 0.0090).

Spirulina increases proliferation/mitochondrial function of human stem cells in culture

We analyzed the effects of spirulina on human neural progenitor cells and human bone marrow and CD34+ cells in culture to determine if spirulina could have a potentially direct effect on the proliferation of stem cells. Using this assay we previously found that NT-020 could promote proliferation of bone marrow and bone marrow derived CD34+ progenitors and progenitor cells from peripheral blood (CD133+) in vitro [14]. Using this previously validated assay, human bone marrow cells were grown in culture with either spirulina alone, NT-020 alone, or spirulina in combination with NT-020 added to the culture media for 72 hours. A significant effect was found in the bone marrow cells proliferation assay (F(5,35) = 166.8, pFig 6.A). Replicating previously published results, NT-020 significantly increased proliferation of bone marrow cells (pFig 6A) [14]. Spirulina alone at concentrations of 62 ng/ml (p