Activated Microglia Inhibit Axonal Growth through RGMa

By causing damage to neural networks, spinal cord injuries (SCI) often result in severe motor and sensory dysfunction. Functional recovery requires axonal regrowth and regeneration of neural network, processes that are quite limited in the adult central nervous system (CNS). Previous work has shown that SCI lesions contain an accumulation of activated microglia, which can have multiple pathophysiological influences. Here, we show that activated microglia inhibit axonal growth via repulsive guidance molecule a (RGMa). We found that microglia activated by lipopolysaccharide (LPS) inhibited neurite outgrowth and induced growth cone collapse of cortical neurons in vitro—a pattern that was only observed when there was direct contact between microglia and neurons. After microglia were activated by LPS, they increased expression of RGMa; however, treatment with RGMa-neutralizing antibodies or transfection of RGMa siRNA attenuated the inhibitory effects of microglia on axonal outgrowth. Furthermore, minocycline, an inhibitor of microglial activation, attenuated the effects of microglia and RGMa expression. Finally, we examined whether these in vitro patterns could also be observed in vivo. Indeed, in a mouse SCI model, minocycline treatment reduced the accumulation of microglia and decreased RGMa expression after SCI, leading to reduced dieback in injured corticospinal tracts. These results suggest that activated microglia play a major role in inhibiting axon regeneration via RGMa in the injured CNS

Sensitivity of turtles to anticoagulant rodenticides: Risk assessment for green sea turtles (Chelonia mydas) in the Ogasawara Islands and comparison of warfarin sensitivity among turtle species

Although anticoagulant rodenticides (ARs) are effectively used for the control of invasive rodents, nontarget species are also frequently exposed to ARs and secondary poisonings occur widely. However, little data is available on the effects of ARs, especially on marine organisms. To evaluate the effects of ARs on marine wildlife, we chose green sea turtles (Chelonia mydas), which are one of the most common marine organisms around the Ogasawara islands, as our primary study species. The sensitivity of these turtles to ARs was assessed using both in vivo and in vitro approaches. We administered 4 mg/kg of warfarin sodium either orally or intravenously to juvenile green sea turtles. The turtles exhibited slow pharmacokinetics, and prolongation of prothrombin time (PT) was observed only with intravenous warfarin administration. We also conducted an in vitro investigation using liver microsomes from green sea turtles, and two other turtle species (softshell turtle and red-eared slider) and rats. The cytochrome P450 metabolic activity in the liver of green sea turtles was lower than in rats. Additionally, vitamin K epoxide reductase (VKOR), which is the target enzyme of ARs, was inhibited by warfarin in the turtles at lower concentration levels than in rats. These data indicate that turtles may be more sensitive to ARs than rats. We expect that these findings will be helpful for sea turtle conservation following accidental AR-broadcast incidents

Activated microglia inhibit neurite outgrowth and induce growth cone collapse in vitro.

<p><i>A</i>, Relative length of neurites of cortical neurons, neurons stimulated by LPS, and neurons co-cultured with BMDM, BMDM stimulated by LPS, microglia, or activated microglia stimulated by LPS (n = 3). <i>B</i>–<i>D</i>, Representative figures of neurons (TuJ1, green, <i>B</i>), neurons co-cultured with unactivated microglia (<i>C</i>), and neurons co-cultured with activated microglia (<i>D</i>). Scale bar: 20 µm. <i>E</i>, Collapse assay (n = 3). <i>F</i>–<i>H</i>, Representative figures of growth cones (phalloidin staining, red) of a neuron (<i>F</i>), a neuron co-cultured with unactivated microglia (<i>G</i>), and a neuron co-cultured with activated microglia (<i>H</i>). Scale bar: 10 µm. Data are represented as mean ± SEM. *<i>p</i><0.01 (one-way ANOVA followed by Tukey-Kramer test).</p

Blockage of micloglial activation by minocycline inhibits CST dieback with the reduction of RGMa expression after SCI.

<p><i>A</i>, Expression of RGMa (green) by CD11b-positive microglia (red) in the lesion epicenter at 7 days after SCI. Scale bar: 20 µm. <i>B</i>, <i>C</i>, Changes in RGMa expression in response to SCI (day 7) and treatment with minocycline (n = 3). *<i>p</i><0.01 (one-way ANOVA followed by Tukey-Kramer test). <i>D</i>–<i>G</i>, Changes in the number of CD11b⁺ cells (red) in an SCI lesion site at 7 (<i>E</i>) and 14 (G) days after treatment with minocycline. These patterns differ from those observed after treatment with the control vehicle only (<i>D</i>, <i>F</i>). Scale bar: 500 µm. <i>H</i>, Results of quantitative analyses investigating CD11b-positive area in minocycline-treated group (M) and vehicle-treated control (C) (7d, C, n = 10; M, n = 8; 14d, C, n = 9; M, n = 9). *<i>p</i><0.05 (Student's <i>t-</i>test). <i>I</i>–<i>L</i>, Comparison of the effects of minocycline administration (<i>J</i>, <i>L</i>) and control vehicle treatment (<i>I</i>, <i>K</i>) on CST dieback at 7 and 14 days, respectively. CST axons (BDA, green) and the accumulating microglial cells (CD11b, red). White line indicates the margin of lesion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025234#s2" target="_blank">Materials and methods</a>). Scale bar: 100 µm. <i>M</i>, Quantitative analyses of CST dieback by minocycline treatment (M) and vehicle-treated control (C) (7d, C, n = 10; M, n = 8; 14d, C, n = 9; M, n = 9). Data are represented as mean ± SEM. *<i>p</i><0.05 (Student's <i>t-</i>test).</p

Cell-cell contact is necessary for activated microglia-induced neurite outgrowth inhibition and growth cone collapse.

<p><b><i>A</i></b>, Results of neurite outgrowth assay across 4 treatment groups (neurons-only, neurons + LPS, neurons co-cultured with unactivated microglia, neurons co-cultured with activated microglia) in the transwell system (n = 3). The neurons, seeded to the lower plane, were separately co-cultured with the unactivated and activated primary microglia, seeded to the upper plane. <b><i>B</i></b>, Results of growth cone collapse assay across 4 treatment groups (neurons-only, neurons + LPS, neurons co-cultured with unactivated or activated microglia) in the transwell system (n = 3). Data are represented as mean ± SEM.</p

RGMa mediates the effect of activated microglia in inhibiting neurite outgrowth.

<p><i>A</i>, Results of Western blot for RGMa in microglia. RGMa expression was higher in activated microglia (LPS) than in unactivated (control) or activated microglia treated with minocycline (LPS + mino). <i>B</i>, Quantitative data for <i>A</i> (n = 3). <i>C</i>, Effect of treatment with anti-RGMa antibody on the neurite outgrowth-inhibiting effects of activated microglia (n = 3). <i>D</i>, Effect of treatment with anti-RGMa antibody on activated microglia-induced growth cone collapse (n = 3). <i>E</i>, Results of Western blot for RGMa in RGMa siRNA- and control siRNA-transfected microglia stimulated by LPS. <i>F</i>, Quantitative data for <i>E</i> (n = 3). <i>G</i>, Relative length of neurites of cortical neurons and that co-cultured with microglia, activated microglia stimulated by LPS, activated microglia transfected with control siRNA (control siRNA), or activated microglia transfected with RGMa siRNA (RGMa siRNA) (n = 3). <i>H</i>, Collapse assay (n = 3). <i>I</i>, Results of Western blot for neogenin in neogenin siRNA- and control siRNA-transfected cortical neurons. <i>J</i>, Quantitative data for <i>I</i> (n = 3). <i>K</i>, Relative length of neurites of cortical neurons transfected with control siRNA or neogenin siRNA, and that co-cultured with activated microglia stimulated by LPS (n = 3). <i>L</i>, Collapse assay (n = 3). <i>M</i>, <i>N</i>, Effect of minocycline on activated microglia-induced neurite outgrowth inhibition (<i>M</i>, n = 3) and growth cone collapse (<i>N</i>, n = 3). Data are represented as mean ± SEM. *<i>p</i><0.01 (<i>B</i>, <i>C</i>, <i>D</i>, <i>G</i>, <i>H</i>, <i>K</i>, <i>L</i>, <i>M</i>, <i>N</i>, one-way ANOVA followed by Tukey-Kramer test; <i>F</i>, <i>J</i>, Student's <i>t-</i>test).</p

Transected axons do not regrow into the lesion of SCI where microglia/macrophages accumulate.

<p><i>A</i>, <i>B</i>, Immunostaining of phospho-neurofilament (SMI31; <i>A</i>; axon, green) and CD11b (<i>B</i>; microglia/macrophage, red) at day 7 after SCI. Scale bar: 500 µm. <i>C</i>–<i>E</i>, BDA-labeled CST fibers (<i>C</i>, green) and CD11b (<i>D</i>; microglia/macrophage, red) at day 7 after SCI. Scale bar: 500 µm.</p