Additional file 3 of Swedish trial on embolization of middle meningeal artery versus surgical evacuation in chronic subdural hematoma (SWEMMA)—a national 12-month multi-center randomized controlled superiority trial with parallel group assignment, open treatment allocation and blinded clinical outcome assessment

Additional file 3: Appendix 3. Informed consent (English translation)

Additional file 4 of Swedish trial on embolization of middle meningeal artery versus surgical evacuation in chronic subdural hematoma (SWEMMA)—a national 12-month multi-center randomized controlled superiority trial with parallel group assignment, open treatment allocation and blinded clinical outcome assessment

Additional file 4: Appendix 4. Trial participation information (English)

Additional file 1 of Swedish trial on embolization of middle meningeal artery versus surgical evacuation in chronic subdural hematoma (SWEMMA)—a national 12-month multi-center randomized controlled superiority trial with parallel group assignment, open treatment allocation and blinded clinical outcome assessment

Additional file 1: Appendix 1. Ethics approval (Swedish)

Additional file 2 of Swedish trial on embolization of middle meningeal artery versus surgical evacuation in chronic subdural hematoma (SWEMMA)—a national 12-month multi-center randomized controlled superiority trial with parallel group assignment, open treatment allocation and blinded clinical outcome assessment

Additional file 2: Appendix 2. Ethics approval (English translation)

LINGO-1 expression increases during neural stem cell differentiation.

<p>A) Western blot analysis was used to study LINGO-1 expression during NSPC differentiation. Cell lysates from proliferating cells (Day 0) and NSPCs differentiating for 1–9 days was immunoprecipitated with anti-LINGO-1 antibodies (Novartis) and blotted with LINGO-1 ab (Abcam). LINGO-1 is present in both proliferating and differentiated NSPCs, but the expression increases during the differentiation. B) Protein quantification show a 9-fold increase in LINGO-1 expression in cultures differentiated for 9 days compared to proliferating NSPCs (Day 0). Double immunostainings with specific antibodies against LINGO-1 (C–N) and nestin (C–E), βIIItubulin (F–H), GFAP (I–K) or CNPase (L–N) show that NSPCs, neurons and oligodendrocytes express LINGO-1, but not astrocytes. The NSPC cultures were fixed at day 0 (C–E) and the differentiated cultures were fixed at day 6 after mitogen withdrawal (F–N). Scale bars = 20 µm.</p

LINGO-1 neutralization promotes cell proliferation.

<p>BrdU labeling was used to investigate the effect of LINGO-1 neutralization on cell proliferation (A–D). Cells were exposed to BrdU for 16 hours prior to fixation and stained with specific antibodies against BrdU (red) and DAPI (blue). E) The number of cells that had incorporated BrdU was counted and plotted as the ratio of BrdU-positive cells to the total cell count. F) The total cell number in anti-LINGO-1 antibody treated and untreated control cultures were measured at day 0, 1, 3 and 6 days of differentiation. The cells in each dish were harvested using a cell scraper and counted using a NucleoCounter™. The mean values of the total cell count/dish were plotted. *** denotes p<0,001, ** denotes p<0,01 and * denotes p<0,05 and scale bars = 20 µm.</p

Neutralization of LINGO-1 leads to an increased percentage of proliferating neuroblasts.

<p>To investigate if the immature neurons in LINGO-1 neutralized cultures proliferate, we double-labeled the cells with specific antibodies against BrdU (red), βIIItubulin (green) and DAPI (blue). NSPCs were fixed at day 0 (A–B) or differentiated in only medium for 3 days (C–D) or for 6 days (E–F) medium containing anti-LINGO-1 antibodies (G–J). BrdU was added to the cultures 16 hours prior to fixation. Scale bars = 20 µm.</p

LINGO-1 neutralization during NSCP differentiation results in immature neurons.

<p>To investigate the effect of LINGO-1 neutralization on NSPC differentiation, NSPCs were cultured for 6 days in the absence (A, C and E) or presence (B, D and F) of LINGO-1 ab (Novartis) following mitogen withdrawal. The cells were fixed and stained with specific antibodies against βIIItubulin (A–B), GFAP (C–D) or CNPase (E–F). In control cultures (A) neurons were rather mature with multiple, long extending processes, but in cultures treated with LINGO-1 antibodies (B) the neurons had a more immature phenotype with only one or two short processes. There was no distinct difference in astrocyte staining (C–D) or oligodrocyte staining (E–F) between control cultures and LINGO-1 inhibited cultures. Scale bars = 20 µm.</p

LINGO-1 neutralization has no effect on PKB/c-Akt phosphorylation.

<p>A) Western blot analysis was used to study PKB/c-Akt phosphorylation in differentiating NSPCs in the absence or presence of LINGO-1 ab. Total cell lysates were used for immunoblotting with anti-PKB/c-Akt antibody (Akt) and anti-phosphorylated PKB/c-Akt antibody (p-Akt). B) The ratio of phosphorylated Akt was measured and plotted.</p

Decreased number of TUNEL positive cells in cultures treated with anti-LINGO-1 antibodies.

<p>TUNEL assay was performed on NSPC and parallel cultures of cells differentiated for 1 and 3 days in the absence or presence of LINGO-1 ab (A–D). Representative photos of control cultures (A–B) and LINGO-1 neutralized cultures (C–D) at day 3 of differentiation, TUNEL (green) and DAPI (blue). E) Cells going through apoptosis were counted and plotted as the ratio of the total number of cells. *** denotes p<0,001, ** denotes p<0,01 and * denotes p<0,05 and scale bars = 20 µm.</p