Fig 1.
(A) Stretch injury microfluidic device. Cell bodies or soma are isolated in the soma compartment and axons extend into the axon compartment through microgrooves (10 μm width and 3 μm high). A gas pressure is applied into the pneumatic channel and deforms the thin PDMS membrane, causing the stretching of axons growing on top. (B) Timeline for the control, very mild (0.5%), mild (5%) and repetitive very mild (2×0.5%) stretch injury experiments. (i) For the control, neurons were seeded and grew on the stretch injury microfluidic device without applying any gas pressure, and fixed and imaged at both DIV 8 and DIV 10. (ii), (iii) Isolated axons were stretched at 0.5% strain or 5% strain at DIV 7 and fixed and imaged at both DIV 8 and DIV 10. (iv) For the repetitive injury investigation, isolated axons initially received 0.5% stretch on DIV 7. 24 h later, a second 0.5% stretch injury was applied on the same device and then fixed at DIV 10.
Fig 2.
Double immunolabelling verified extensive network of axons (βIII tubulin immunoreactivity) and growth cones (F-actin staining) within the axon compartment of the stretch injury model at 7 DIV.
(A) Axons (green; βIII tubulin) of primary rat cortical neurons extended into the axon compartment through microgrooves (450 μm long, 10 μm width and 3 μm high) at 7 DIV. Dashed lines indicate the location of the pneumatic channel (or stretch injury) and solid lines show the microgrooves region. (B) High magnification of axons with growth cones immunostained with F-actin (red) and microtubules (green) (white square box in panel A). Insets show higher magnification of growth cone. Scale Bars = 200 μm (A), 75 μm (B), 40 μm (insets).
Fig 3.
Graphs showing average growth cone area of control, 0.5% stretched, 5% stretched and repetitive very mild (2×0.5%) stretched axons at different time point.
There was a significant decrease in growth cone size after single stretch injury at both 24 h and 72 h PI and after repetitive very mild (2×0.5%) stretch injury at 72 h PI compared to control. *p<0.05. Error bar = mean ± SEM.
Fig 4.
Immunocytochemistry images of growth cones of control (uninjured), 0.5% stretched and 5% stretched axons at 10 DIV and 72 h PI.
(A-C) In the control, growth cones with distinct filopodia were apparent (arrow). The βIII tubulin labelling (green) was distributed predominantly within the central domain of the growth cone while F actin (red) was confined to the peripheral region. (D-F) In the 0.5% stretched axon, microtubules appeared to form a loop in the central region of the growth cones and F-actin was confined to the peripheral and transition region only. (G-I) In the 5% stretched axon, F-actin was most abundant in the axon tip, forming bulb like accumulations. Scale Bars = 10 μm.
Fig 5.
Graphs showing the mean percentages of collapse growth cone and the extent of colocalizatuon of F actin and βIII tubulin in growth cones of control cultures and cultures after 0.5% stretched, 5% stretched and repetitive very mild (2×0.5%) stretched axons at different time point.
(A) Stretch injury induced increased axonal growth cone collapsed at both 24 h and 72 h PI compared to the control. In addition, repetitive very mild (2×0.5%) stretch injury induced more collapsed growth cones when compared to single 0.5% stretched axon at 72 h PI. (B) The growth cones in 5% stretched axon had significantly higher colocalization value of βIII tubulin and F-actin compared to both the growth cones in control and 0.5% stretched axon at 72 h PI. However, there was no significant difference between the growth cones in control, 0.5% stretched or 5% stretched axon at 24 h PI. The growth cones in 2×0.5% repetitive stretched axon has significantly higher colocalization value of βIII tubulin and F-actin if compare to both the growth cones in control and single 0.5% stretched axon at 72 h PI. *p<0.05. Error bar = mean ± SEM.
Fig 6.
Tau (microtubule marker) immunofluorescence labeling demonstrating the effect of EpoD exposure on 5% stretched axons at 7 DIV.
(A) Representative fluorescence images of unstretched and stretched culture 24 h after the indicated treatment. (B) EpoD (100 nM) applied to axon compartment alone for 24 h induced a significant decrease in degenerative index in the axon compartment when compared to vehicle- treated cultures following injury. The arterisk indicates the difference from paired control. *p<0.05. Error bar = mean ± SEM. Scale bar = 30 μm.