Larvae exhibit variable locations and sizes of haematoma, and resolution is spontaneous. (A) Representative dorsal view, inverted images of gata1:dsRed fluorescence in a single larva (Fish 7) from 2, 3, 4 and 5 dpf showing haematoma clearance and restoration of circulation. Scale bar = 100 µm. (B) The number of haemorrhages in ICH+ larvae over time (n = 7) **P = 0.0068, F(2.290, 13.74) 2.638 (C) the volume of the haemorrhages **P = 0.0042 *P = 0.0119, F(2.047, 12.28) = 15.23. (D) The surface area of the haemorrhage *P = 0.012, **P = 0.0023, ##P = 0.0024, F(1.934, 11.60) = 21.27 and (E) the surface area to volume ratio of the haemorrhage **P = 0.002, F(1.711, 10.27) = 9.793. Symbol points pair time data for each individual larva. Data were analysed using a repeated measures one-way ANOVA with uncorrected Fishers post hoc analysis to account for no multiple comparisons (F) Representative images of larvae stained for haemoglobin at 2 (n = 24), 3 (n = 28), 4 (n = 45) and 5 (n = 27) dpf shows the distribution of haematoma across the brain changes over time (white arrows). Scale bar = 75 µm. (G) Percentage of larvae with visible haemorrhage over time. Analysis generated from data in (F). (H) Location of bleeds over time as scored from larvae in (F). dpf, days post-fertilisation; ICH, intracerebral haemorrhage; ns, non-significant; Pro, prosencephalon; mes, mesencephalon; rhom, rhombencephalon.

Live time lapse imaging shows RBCs moving through the ventricular system. A live fli1:EGFP;gata1:DsRed (endothelial cells, RBCs) transgenic larva were imaged over time to obtain a time lapse video (Video 1). (A) Still image from Video 1 (with false colour overlay) showing the location from the magnified insets. A gata1 positive RBC, denoted by a star, is observed in the hindbrain tissue and moves over the course of six frames (∼30 min) into the cluster of RBCs observed in the hindbrain ventricle. Scale bar = 100 µm. (B) Tracks of each gata1 positive cell over time show an overall migration to the hindbrain ventricle and movement within the haematoma. (C) The average automated detection quality was similar between the different cell tracks, one data point per cell. (D) Total number of cells identified (spots) decreased over the course of the time-lapse with the death of RBCs and the loss of gata1 fluorescence. (E) Average X movement versus average Y movement from each cell (spot) shows a high density of tracks migrating to the bottom right of the image, where the hindbrain ventricle is located.

The spontaneous recovery of brain injury after ICH is associated with increased nestin expression. (A) Kaplan Meier analysis shows no difference in survival between WT, ICH− and ICH+ larvae at 21 dpf (93% ICH− and WT, 92% ICH+, N = 35 larvae per group at 5 dpf). (B) Representative images of annexinV expression in an ICH+ larvae at 2, 3, 4 and 5 dpf show apoptotic cells within the head during early development. A dense cluster of dying cells associated with ICH (denoted by white dotted line) at 3 dpf (24 h post-ICH) is resolved by 4 dpf. Scale bar = 100 µm. (C) Representative images of nestin positive larvae from both ICH− and ICH+ groups at 2–5 dpf. Global expression of nestin in the developing larval brain is lost over time with the greatest reduction in expression between 2 and 3 dpf. Overall, ICH+ larvae show a greater intensity of nestin expression. Yellow arrow denotes the absence of nestin expression between hemispheres and white arrows denote regions of intense expression of interest. Scale bar = 100 µm (n = 20, n = 12 at 5 dpf). (D) Quantification of the intensity fluorescence of nestin signal in the brain (n = 12–20). Data were analysed using a one-way ANOVA, **P = 0.0063, ***P = 0.0003, F(3,71) = 19.12. (E) Comparison of nestin expression in the brain at 3 and 4 dpf between ICH− and ICH+ larvae. Data were analysed using a one-way ANOVA ****P < 0.0001, F(3, 110) = 100.9. (F) Paired 3 and 4 dpf data show no difference in brain region of nestin expression. ICH−, intracerebral haemorrhage negative; ICH+, intracerebral haemorrhage positive; WT, wildtype; dpf, days post-fertilisation; ns, non-significant; ALLG, anterior lateral line ganglion; MHB, mid-hindbrain boundary.

Nestin positive cells are more proliferative in ICH+ than in ICH− siblings. (A) Representative western blots and quantification analysis (n = 3) suggests that ICH+ larvae have less neuN-positive mature neurons than ICH− siblings, but pcna-positive proliferative cells and olig2-positive oligodendrocytes are increased in haemorrhage conditions (SD). Data analysed using a two-way ANOVA and no significant differences were found. Uncropped blots Supplementary Fig. 4. (B) Mass spec analysis of up (green) and down (pink) regulated pathways in ICH+ larvae between 3 and 4 dpf; dpf, days post-fertilisation.