Axotomy and regeneration of adult zebrafish RGCs at DIV 3–4. Representative confocal live cell microscopy images taken before, upon and after injury in the same section of the AC, disclose that adult zebrafish RGCs regenerate spontaneously upon in vitro axotomy in SOC450 MFDs. (A) At DIV3, when numerous adult Tg(Tru.gap43:GFP)mil1 (gap43) zebrafish RGC axons are growing in the AC (left panels), an axotomy is performed using dual aspiration of all medium in the AC (DIV 3, 0 hpi, middle panels). One day later, at DIV 4 (24 hpi), numerous regenerating axons can be observed in the AC (regenerating axons, green arrowheads in right panels). However, some axons do not regenerate and stay stationary after axotomy (static axons, red arrowheads) or even degenerate upon injury (degenerating axons, blue arrowheads). Lastly, some axons, that did not reach the AC at the time of axotomy, further extend and form newly outgrowing axons that reach the AC in the hours following axotomy (0 hpi-24 hpi) (newly outgrowing axons, yellow arrowheads). These different states of axons can be distinguished using overview pictures at DIV 3 and DIV 4 in combination with time-lapse live imaging (Supplementary Movies 1, 5). (B) Representative live cell images taken at different time points after axotomy in the same section of the AC of a gap43 retinal culture after axotomy at DIV 3 reveal that regeneration is a dynamic process, and different axons extend at different moments and with different growth rates. Some pioneering axons start to regenerate immediately upon injury (0–1 hpi, *), while others only emerge after a few hours in the AC (1–3 hpi, ×). Of note, occasionally axons make a U-turn and grow back into the microgrooves, rather than regrowing into the AC (purple arrowhead). Scale bar: 100 μm. AC, axonal compartment; DIV, days in vitro; hpi, hours post injury; MFD, microfluidic device; RGC, retinal ganglion cell.
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