ICH+ brain hemorrhage phenotypes. Examples of larval ICH phenotypes maintained on a transgenic gata1:DsRed reporter nacre background observed with a brightfield stereomicroscope (top panels) and fluorescence (bottom panel) at ~48 h post-fertilization. No hemorrhages were observed in ICH-larvae (left panels). A distinct accumulation of red blood cells in the forebrain and hindbrain (arrows) were observed in ICH+ larvae (right panels). Scale bars represent 250 µm. Figure has been reproduced from Crilly et al.²⁰ with permission under a Creative Commons license.

Intracerebral hemorrhage (ICH) in zebrafish larvae results in a quantifiable brain injury. (A) Representative images of the brain injury phenotype in ICH+ larvae (right panels), in comparison to ICH- siblings (left panels), at 72 hpf. Brightfield images (bottom panels, scale bar = 250 µm) demonstrate the presence of brain bleeds (arrows) in ICH+ larvae. Fluorescent microscopy was performed to visualize cell death in the ubiq:secAnnexinV-mVenus reporter line (top panels, scale bar = 100 µm). Clusters of dying cells were observed in peri-hematomal regions. Images were cropped to brain-only regions and analyzed for total green fluorescence intensity in round particles larger than 30 pixels in diameter (white line) using the macro in Supplementary File 1. (B) Quantification of fluorescence signal in the brains of untreated, ICH- and ICH+ larvae obtained through the ATV model (n = 12 per group; 3 independent replicates) at 72 hpf. Significant differences were observed when comparing ICH+ with untreated (**p = 0.004) and with ICH- (*p = 0.03) siblings. (C) Quantification of fluorescence signal as a read out for annexinV binding in the brains of ICH- and ICH+ larvae obtained through the bubblehead (bbh) model (n = 12 per group; 2 independent replicates) at 72 hpf. Graphs show SD from the mean. A significant difference in mVenus fluorescence was observed between ICH+ and ICH- age-matched siblings (**p = 0.002). Figure has been reproduced from Crilly et al.²⁰with permission under a Creative Commons license.