Four- to six-week-old mice were used to isolate the primary hepatocytes

okine release. In the case of ctrlp0 microglia, the release of all tested cytokines and chemokines was significantly reduced by the application of 100 mM MB, the release of TNFa, IL-6 and IL-12 being already significantly reduced by the application of 40 mM MB and that of IL-6 and IL-12 by 10 and 20 mM MB. Application of 1 mM MB had no significant SCH58261 effects on cytokine and chemokine release. Delayed onset of disease in MB-treated SOD1G93A mice MB has been used for a long time in various areas of biology and medicine, e. g. as first line treatment of methemoglobinemia and recently beneficial effects of MB have been reported for clinical trials in AD. To test whether MB has a beneficial effect on mSOD1 disease course, they were orally treated with MB added to the drinking water. Given the knowledge about the well-studied and safe drug which has been also administered orally in human as well as about its high bioavailability, the oral route was chosen for MB administration. Whereas treatment with 3 or 10 mg MB significantly delayed the onset of disease compared to controls, treatment with 30 or 100 mg had no significant effect on disease onset . Correspondingly, the onset of weight loss was delayed in mice treated with 3 or 10 mg MB, but weight loss was not itself significantly improved by MB. The progression to the later clinical stages was only delayed in the 3 mg group by oral MB-treatment Control and MB-treated SOD1G93A mice with respect to the different stages of disease. Weight and survival profiles for SOD1G93A mice. In B the data are presented relative to the highest value in each group. Values are presented as mean 6 SEM; ctrl: n = 16 mice, 3 mg: n = 12, 10 mg: n = 10, 30 mg: n = 8, 100 mg: n = 11; ANOVA followed by Tukey test. Methylene Blue and Neuroinflammation in ALS 10 mg: 122.463.85 days, 30 mg: 121.763.39 days, 100 mg: 126.362.05 days). Accordingly, survival time was not significantly altered by oral MB-treatment, even though the death was slightly delayed by the treatment with 3 mg MB and to a lesser extent with 100 mg MB. Effect of systemic application of MB on microglial activation and motor neuron survival To test the influence of systemically applied MB on microglial reaction to laser-induced axon transection within the lateral column and on motor neuron survival, we treated SOD1G93A mice with MB and performed 2P-LSM recordings for microglia activation and immunohistochemistry for quantification of lumbar anterior horn neurons. No significant differences were observed in control versus treated mice when microglial reactions towards single axon transections were studied . Similarly, microglial reactions were comparable in mice at different clinical stages. Accordingly, Iba1 immunostaining of the lumbar anterior horn showed no differences between MB-treated SOD1G93A mice and controls with respect to microgliosis. To determine whether MB-induced increases in motor neuron survival could underlie the observed delay of disease onset, NeuNstained cross-sections of the spinal cord at level L3 to L5 were investigated. Pre-clinically, the number of counted neurons was significantly higher in MB-treated mice than in control siblings . However, this difference in neuron count was not present at the clinical and advanced clinical stages indicating only an initial and temporary effect of MB SOD1G93A mice. The time until mice fell off the rotarod at 12 rpm and the velocity the mice reached using an acceleration rate of 1 rpm every 10 s