Fig. 3
A single CSD triggered by pinprick causes release of HMGB1 from neurons within small EVs, which are subsequently taken up by astrocyte processes. (A) Immunolabeling reveals numerous HMGB1-positive puncta (red, marked by white arrows) in the cytoplasm surrounding the nuclei of cortical neurons, identified by CD171 immunolabeling (green). Insets below delineate the boundaries of neuronal cytoplasm and nucleus, emphasizing the distribution of the puncta. Shedding of HMGB1-labeled puncta from cells, with varying degrees of nuclear HMGB1 immunopositivity loss, is observed as early as 15 min post-CSD. Puncta near the nuclei (white arrows) suggest HMGB1 release within vesicles. Images are maximum projections of confocal z-stacks. Scale bars: 10 μm. (B) Electron microscopic (EM) images of a neuron depict a multivesicular body (light blue) containing several small EVs, one of which carries gold nanoparticles marking HMGB1 1-hour post-CSD. (C) Transmission EM image of EV suspension isolated from mouse brain, predominantly having a size compatible with exosomes. (D) 3D surface reconstruction of a GFP‐positive astrocyte and its process shows that HMGB1-immunopositive puncta (black triangles) are located inside the process. The black rectangle on the left panel indicates the HMGB1‐immunopositive process that is visualized on the right panels from different angles in 3D. P and D denote the proximal and distal ends of the process, respectively. Scale bars: 2 μm. X, Y, and Z axes of the volume are shown for orientation. Reproduced from [41] under Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/)