Inhibition of microglial cytokines and chemokines production by MB.

<p>Primary cultures of control (newborn control: ctrl_p0; adult control from corresponding siblings of mutant mice: ctrl_p120) or mutant (from advanced clinical SOD1^G93A mice; G93A_p120) microglia (15,000 cells/well) were incubated with MB (1, 10, 20, 40 and 100 µM) in the presence or absence of LPS (100 ng/ml) for 18 hours. Cytokine and chemokine release profile was determined in the supernatants. Data are presented relative to the corresponding value of LPS-triggered cytokine or chemokine release from control microglia of newborn mice (set as 1). Data are mean ± SEM of triplicates of 3 (MCP-1), 4 (IL-6) or 5 (TNF-α, RANTES, KC, MIP-1α and IL-12) independent experiments; ANOVA followed by Tukey test (*p<0.05, **p<0.01, ***p<0.001).</p

Inhibition of microglial reaction towards axonal injury in the lateral column by local application of MB <i>in vivo.</i>

<p>Microglial reaction towards laser-induced axonal transections within the lateral column of the spinal cord was recorded. Tissue injuries were induced by high-power laser pulses. The experiments have been performed in double transgenic mice expressing EGFP in microglia and EYFP in projection neurons. For better visualization, EYFP fluorescence in the images is depicted with a red colour table. Images are arranged such that rostral is to the upper side. (A-D) Left images were taken immediately (3 min) after axonal transection (autofluorescence and arrow) in control and mutant (SOD1^G93A) mice. Respective images (right) were taken 60 min after injury. In respective experiments spinal cord was superfused with MB (1 mM). (E) Quantification of microglial response (increase of EGFP fluorescence around the injury) to the injured site. Control and mutant mice were of corresponding age (60 to 90 days of age). n = 7 mice for control response, n = 4 for MB-modified response in control mice, n = 9 for response in SOD1^G93A mice, n = 7 for MB-modified response in SOD1^G93A mice. (F) Breeding strategy to obtain SOD1^G93A mice with fluorescently labeled microglia and projection neurons. Values are presented as mean ± SEM; ANOVA followed by Tukey test (*p<0.05, **p<0.01, ***p<0.001).</p

Delayed onset of disease by oral application of MB.

<p>(A) Control and MB-treated (3, 10, 30 and 100 mg oral per kg body weight per day) SOD1^G93A mice with respect to the different stages of disease (stage 1: onset of disease/early clinical stage; stage 2: clinical stage; stage 3: advanced clinical stage). (B and C) Weight and survival profiles for SOD1^G93A mice (control and MB-treated). In B the data are presented relative to the highest value in each group. Values are presented as mean ± 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 (*p<0.05, **p<0.01).</p

Effect of systemic application of MB on intracellular TDP-43-containing aggregates.

<p>To analyze intracellular aggregations in anterior horn neurons, TDP-43 and SMI-32 staining was performed in lumbar cross-sections. TDP-43-containing aggregates in SMI-32-positive neurons were observed in the anterior horn of control and MB-treated pre-clinical and advanced clinical mice. An exemplary TDP-43-stained neuron from each group, marked by an arrow, is shown in the last line of images.</p

Delayed motor disturbance by MB application.

<p>Rotarod motor performance of untreated and MB-treated (10 mg intraperitoneal injection per kg body weight per day) SOD1^G93A mice. The time until mice fell off the rotarod at 12 rpm (A) and the velocity the mice reached using an acceleration rate of 1 rpm every 10 s (B) are presented. Each animal was tested three times per trial. Values are presented as mean ± SEM; both groups: n = 10 mice; ANOVA followed by Tukey test (*p<0.05, **p<0.01).</p

Delayed onset of disease by intraperitoneal application of MB.

<p>(A) Control and MB-treated (10 mg intraperitoneal injection per kg body weight per day) SOD1^G93A mice with respect to the different stages of disease. (B and C) Weight and survival profiles for SOD1^G93A mice (control and MB-treated). In B the data are presented relative to the highest value in each group. Values are presented as mean ± SEM; both groups: n = 16 mice; ANOVA followed by Tukey test (*p<0.05, ***p<0.001).</p

Effect of systemic application of MB on motor neuron survival.

<p>(A) NeuN-stained cross-sections of the spinal cord at the level L3 to L5. Non-treated controls (SOD1^G93A mice) are depicted left. Corresponding sections of MB-treated SOD1^G93A mice are shown on the right (10 mg oral per kg body weight per day; drug administration started at the age of 45 days) (B) Counting of neurons in the anterior horn of SOD1^G93A mice at different disease stages. Cell somata bigger than 20 µm were counted and given per mm². In preclinical stages, neuron number was significantly higher in MB-treated mice compared to non-treated mice indicating an early neuroprotective effect of MB. Note that no differences were observed in later disease stages. Values are presented as mean ± SEM; n = 4 mice for each group; ANOVA followed by Tukey test (*p<0.05).</p

Effect of systemic application of MB on microgliosis.

<p>Iba1 staining of the anterior horn at level L3-L5 during the disease course of SOD1^G93A. MB-treatment did not inhibit the observed microgliosis. Arrows point to the central canal.</p