JNK1 2/2 mice displayed similar ratios of CD4, CD8, cdT, and NKT cells as WT mice

are presented. Each animal was tested three times per trial. Values are presented as mean 6 SEM; both groups: n = 10 mice; ANOVA followed by Tukey test. doi:10.1371/journal.pone.0043963.g005 8 Methylene Blue and Neuroinflammation in ALS 68.265.59 per mm2, MB: 75.866.71; advanced clinical stage at 130-day-old mice: ctrl: 36.563.2, MB: 36.466.25). To analyze intracellular aggregations in affected motor neurons, TDP-43 and SOD1 staining was performed in lumbar cross-sections. Discussion Neuroinflammation is a prominent feature of murine ALS animal models. Despite strong evidence for microglial activation and release of pro-inflammatory cytokines driving disease progression, anti-inflammatory strategies have been overall disappointing. Our earlier findings using in vivo time-lapse imaging of the spinal cord 6dorsal tracts indicated that the NOS and sGC inhibitor Methylene Blue and Neuroinflammation in ALS 10 Methylene Blue and Neuroinflammation in ALS methylene blue strongly reduce microglial activation by inhibiting NO-mediated reactions towards tissue injury. The present study confirmed this for the lateral columns of the spinal cord in both control and mutant mice. This region contains efferent fibers that undergo degeneration in ALS. Furthermore, addition to this local anti-inflammatory effect in vivo, MB at high concentration significantly inhibited the induction of cytokines and chemokines in control and mutant microglia in vitro. These results prompted us to study potential therapeutic effects of MB in an ALS mouse model. MB influenced the course of the disease in SOD1G93A mice when administered from preclinical stages onwards via oral or intraperitoneal routes. For both MB significantly delayed the onset of disease by nearly 10 days when, in the case of oral application, it was administered at 3 or 10 mg per kg body weight per day. The Time from onset of disease to death was not changed in either group indicating that overall disease progression was not affected. In confirmation, a recent study in which 1 or 10 mg MB per kg body weight per day was administered intraperitoneally beginning at the onset of SOD1G93A and TDP-43G348C disease showed no extension of the lifespan. Thus, neuroprotection by MB seems to be more crucial in preclinical than in clinical stages. A further increase of MB dose in our trial had no effect on disease course which was unexpected in view of the previously observed local anti-inflammatory effects after application of MB at high doses in vivo and in vitro. Administration at 30 or 100 mg per kg body weight per day from preclinical stages onwards had no significant effects on onset or survival, as has been recently shown by a treatment trial in which 25 mg per kg body weight per day was administered orally beginning at a similar preclinical stage. Apparently, the high concentrations needed inside and locally within the central nervous tissue for anti-inflammatory effects cannot be achieved by increasing the dose of systematically administered MB. Moreover, increasing the dose actually led to a loss of the Rutoside price beneficial neuroprotective effects of MB when administered at 3 or 10 mg per kg body weight per day. The reason for this neutralization is unclear. Similar loss of beneficial effects has been shown, for instance, for imipramine in mdx mice. As known from human clinical trials, the dose of a drug has to be optimized as an increase may abolish beneficial effects, due, for instance, by inducing unwanted side e