Fig. 1

Set-up of a novel axonal injury system. A Schematic representation of the injury system. Image of microfluidic chamber compartments with WGA-stained neurons (scale bar: 500 µm). B Quantification of the speed of the media at different pump flow rates by tracking fluorescent beads in the flow channel (n = 3 independent chambers). C Calculation of the maximum stress applied in the flow channel by quantification of the urethane pillar bending (n = 3 independent chambers, scale bar: 20 µm, close-up: 3 µm). D and E Flow cytometry quantification (D) and immunofluorescence images (E) of the expression and localization of neuronal lineage markers through the differentiation stages from NSCs to mature neurons (FACS: n = 3, 250.000 cells/n; IF: n = 3, scale bar: 100 µm). F Electrophysiological activity during neuronal terminal differentiation (from DIV0 to DIV90, expressed in weeks) recorded on MEA plates. Right plots: representative raster plots of DIV40 and DIV76 recordings (n = 3, 4 wells/n). G Quantification of Ca²⁺ transients in cultures incubated with Fluo-4 AM through neuronal terminal differentiation (from DIV0 to DIV40) (n = 2, 5 recordings/n, scale bar: 50 µm). H Immunostaining against Tau, β3-Tub, Map2 and pNF (SMI31) of DIV40 neurons cultured in microfluidic chambers. Axonal and dendritic length measurements from the neuronal compartment to the axonal compartment (n = 3, 5–10 projections/n, scale bar: 200 µm). I Effects of different pump flow rates on AS generation and axotomy. Axons of mem-mCherry transduced neurons (DIV40) imaged before and after different pump flow rates for 90 s (n = 5, scale bar: 10 µm). TEM of axons in the flow channel (scale bar: 2 µm). Data are mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001). Statistical comparisons were performed using one-way ANOVA followed by Dunnett’s multiple comparison test against NSCs group (D) or DIV10 group (G), or the Tukey’s multiple comparison test (H and I). See also Additional file 1: Fig. S1