Figure 6

Axotomy and regeneration of repeatedly injured adult zebrafish RGCs at DIV 3–5. Adult zebrafish RGC axons that have successfully sprouted after a first in vitro axotomy, regenerate again when a second in vitro axotomy is executed. Representative confocal live cell microscopy images taken before, upon and after injury in the same section of the AC of cultured Tg(Tru.gap43:GFP)^mil1(gap43) retinal neurons in an open compartment (SOC450) MFD disclose that 24 h after both the first (DIV 4, 24 hpi, 3th panels) and the second axotomy (DIV 5, 24 hpi2, 5th panels), regenerating (green arrowheads) as well as newly outgrowing (yellow arrowheads), static (red arrowheads) and degenerating axons (blue arrowheads) can be observed in the AC. These results indicate that repeated injuries (in vivo ONC, isolation, in vitro axotomy) do not negatively impact the spontaneous regenerative potential of cultured zebrafish RGCs. Of note, the high number of outgrowing and regenerating axons at DIV 5 complicates the correct identification of individual axons in the AC. Therefore, no further analyses should be performed on these cultures. Scale bar: 100 μm. AC, axonal compartment; DIV, days in vitro; hpi, hours post injury; MFD, microfluidic device; RGC, retinal ganglion cell.