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Microglial invasion in the neuropil correlates with the kinetics of wound closure. (A) Displacement field analysis showing the direction of displacement of individual neuronal nuclei (black arrows). The orange lines show how the intersection points (black spots) between trajectories are estimated. (B) Polar representation (in degrees) of the position of intersection points of individual trajectories of neuronal nuclei from 10 animals after tectum size normalisation and rotation. The optic tectum is represented as an overlay (boundaries in dark grey and PVZ in light grey). (C) Accumulation of cells in the neuropil (inside the orange circle) after injury in Tg(h2a:GFP) larvae at 20 hpi. (D) Identification of the accumulated cells as mpeg1+ cells using Tg(mpeg1:mCherry) (red) larvae, combined with nuclear staining (Hoechst 33342, grey). (E) Identification of the mpeg1:GFP+ cells as microglia using 4C4 immunofluorescence in an injured Tg(Xla.Tubb:DsRed); Tg(mpeg1:GFP) fish. White arrows point out mpeg1:GFP+/4C4+ double-labelled cells. (F) Polar representation of microglial positions (N = 308 from 15 animals, magenta) in the neuropil compared with intersection points of trajectories of neuronal nuclei (black, green spots: centre of gravity of intersection points for each animal). (G) Selected frames of a time-lapse imaging series of an injured Tg(Xla.Tubb:DsRed); Tg(mpeg1:GFP) transgenic animal show the recruitment and accumulation of microglia in the injury site. (H) Lower magnification image of an injured Tg(Xla.Tubb:DsRed); Tg(mpeg1:GFP) transgenic animal showing that microglia did not accumulate in the intact contralateral tectum. (I) Kinetics of microglial accumulation from 2 hpi to 13 hpi (black square) and the average wound closure kinetics (black dots). (J) Dual-channel single-cell tracking of neuronal cell bodies (left, red trajectories) and microglia (right, green trajectories) in an injured Tg(Xla.Tubb:DsRed); Tg(mpeg1:GFP) larva. (K) Boxplot representation of the discrete Fréchet distance estimation between microglial trajectories and experimental (orange) and randomised (blue) trajectories of neuronal cell nuclei. Box plots show the median, box edges represent the 25th and 75th percentiles, and whiskers show ± 1.5 x the interquartile range. t test, P <0.05. Scale bars represent 50 μm on all images.
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