Fig. 6.

Activity of label-retaining cells in limbal epithelia 0–24 h after corneal wounding. (A) Schematic representation of a pulse-chase-pulse experimental design illustrating the exposure of 8 week-old C57BL/6 mice to 30 days IdU treatment, subsequent 11-weeks washout, CldU injection at 0 h after unilateral corneal epithelial wound in vivo, and mice death at 24 h. (B) Immunofluorescent images of proliferative cells in flat-mounted idU-labelled limbal epithelium after wounding and CldU pulse: (b1) IdU (red); (b2) CldU (green); (b3) Merged image including TO-PRO3-label (blue). Double-labelled cell indicated by white arrows are actively proliferative label-retaining cells. Yellow arrows indicate IdU-positive, CldU-negative (stem) cells that did not divide after wounding. (C) Cell proliferation in the limbal epithelia of unwounded and wounded corneas. Data are expressed as mean ± SEM of: %IdU-positive cells as a proportion of all cells in limbal epithelium – pink/red), %CldU-positive cells (green), and % of double-labelled cells (yellow) 24 h after wounding. Grey bars show the % of double-labelled cells as a proportion of the IdU-positive cells and represents the percentage of label-retaining stem cells that were dividing in wounded and unwounded eyes. There was a significant sevenfold increase in the percentage of label-retaining stem cells entering mitosis in wounded eyes (P < 0.05 (*), Mann–Whitney U test, n = 6 wounded, 6 unwounded. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)