Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation

Abstract

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

“…Although for sample preparation relatively standard EM processing (fixation, embedding and sectioning) is needed [5][6][7] , it is technically challenging to cut large ultrathin sections completely devoid of artifacts. Sections are very fragile, easily break, fold or are destroyed during imaging, which typically takes multiple hours per dataset.…”

“…By large-scale EM areas up to square millimeters can be visualized. We and others previously applied nanotomy to rat pancreatic islets 4 , cell culture 2 , rat brain 3 , skin and mucosa 7 and whole zebrafish larvae 1,5 (www.nanotomy.org). Zebrafish are highly suitable for in vivo imaging, in particularly to visualize cell types which are difficult to access in mammalian tissues including brain immune cells 11 .…”

“…2. When processing samples for correlative EM after acquiring in vivo imaging data using confocal or 2 photon imaging in 1.8% agarose as described, 5,15 fix larvae in agarose using 4% PFA in PBS containing Triton-X-100 (0.05%).…”

“…Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae [1][2][3][4][5][6][7] . Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features.…”

“…Large scale EM images are typically ~ 5 -50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture [1][2][3][4][5] . Here, zebrafish brains were analyzed in a noninvasive neuronal ablation model.…”