Astrocytes gather cell corpses and engulf cell debris after scratch injury.

<p>(A) An astrocyte (red A) gathers dead cells (yellow arrowheads) by adhering to the dead cells with its processes' before dragging the dead cells towards the cell body. First picture is taken 40 minutes after injury and time indicated for each picture is time lapsed after the first image. Scale bar = 20 µm, the dashed lines represent the scratch. (B) An astrocyte (red A) actively gathers and engulfs cell debris (yellow arrowhead) that directly after engulfment appears in a vacuole within the astrocyte. First picture is taken 10 h, 40 minutes after injury and time indicated in the pictures is time lapsed after the first image. Scale bar = 10 µm.</p

Astrocytes are ineffective in degrading the engulfed cells.

<p>Apoptotic cells were pre-labeled with pHrodo-dye and added to (A,C) control macrophage cultures and (B,D) neuronal cell cultures. Parallel neural and macrophage cell cultures were fixed after (A–B) 5 h or (C–D) 3 d. (A) After 5 h, macrophages had already started to degrade phagocytosed cells in the lysosomes (bright red intracellular compartments). (B,D) Astrocytes did not contain red fluorescing material at any time point, indicating that the engulfed cell corpses did not fuse with lysosomes. (C) Dead cells ingested by macrophages had been degraded after 3 days and only remnant pHrodo-dye was apparent intracellularly. (D) In contrast to the macrophages, astrocytes had accumulated more intact DAPI labeled nuclei at day 3, but did not fluoresces red, indicating that the ingested cells had not fused with lysosomes at this time. (E) BrdU labeled, apoptotic cells were added to neuronal cultures and parallel cultures were fixed after 1 and 3 days (1 d respective 3 d in graph) or were carefully washed after 1 or 3 days and incubated for an additional 2 days in medium without apoptotic cells (1 d+2 d respective 3 d+2 d in graph) prior to fixation. Counting of the ingested BrdU+ nuclei show that astrocytes continued to accumulate cell corpses during the 1 day to 3 days of incubation, but after 2 days in medium without dead cells the ingested dead cells had still not been degraded. Error bars represent SEM.</p

Molecular mechanisms behind the macropinocytosis-like engulfment in astrocytes.

<p>(A) TEM image of an astrocyte in the process of engulfing a dead cell (white star) show interaction points (black arrowheads) indicative of receptor/ligand interaction. Dead cells were added to the culture and incubated for 22 h before fixation and prepared for TEM. (B) Phagocytic genes are expressed in the neural cultures. qRT-PCR data showing the expression of <i>GFAP</i>, <i>Megf10</i>, <i>Crk</i>, <i>Rac1</i> and <i>Mfge8</i>. The average of 5 independent injured and 5 uninjured neural cultures are presented. <i>Megf10</i>, <i>Crk</i>, <i>Rac1</i> and <i>Megf8</i> were expressed in all the cultures. Error bars represent SEM. (C) The protein levels of MEGF10 is up-regulated after addition of dead cells. Fresh medium with or without dead cells were added to cell cultures, differentiated for 8 days. The cells were incubated for an additional 1 day (9 days in graph) or 3 days (12 days in graph). Cell lysates were analyzed for MEGF10 expression by Western blot and β Tubulin served as a loading control. (D–E) Engulfed cells are found in macropinocytotic-like vacuoles. Twenty-two hours after scratch injury, the cultures were fixed and astrocytes were identified with specific antibodies to GFAP. Phalloidin and DAPI labeling was used in order to visualize the actin cytoskeleton and cell nuclei, respectively. To be counted as engulfed, the dead cells (condensed nuclei) had to be situated in cytoplasmic vacuoles (white arrowheads) within the astrocytes. (F–H) The highly vacuolized astrocytes appear most active in cell corpse clearing. (F–G) The micrographs show a highly vacuolized astrocyte before engulfment (F) and approximately 28 h later the same astrocyte have ingested several dead cells (G). First picture is taken 25 minutes after injury and time indicated in the pictures is time lapsed after the first image. The dashed line represents the cut. (H) The vacuoles (black arrowheads) can be seen in transmission electron microscope as round vesicles, some apparently empty whereas others contain debris. An astrocyte-ingested dead cell is marked by a white star and the nucleus by Nu. Scale bars: 200 nm (A), 10 µm (D–G), 2 µm (H).</p

Engulfing astrocytes are present in the brain following acute injury.

<p>(A) Astrocytes derived from the adult brain engulf cell corpses. Mixed cell cultures derived from SVZs of adult animals were injured and fixed after 22 hours. Dead cells were identified by TUNEL staining (green), astrocytes with specific antibodies against GFAP (red) and nuclei by DAPI (blue). Confocal micrograph shows a dead, TUNEL positive cell within an astrocyte (white arrows). (B–M) Traumatic brain injury in mice elicits astrocytic engulfment of dead cells at the site of injury. Animals that received CCI-injury were perfused after one (<i>n</i> = 5), three (<i>n</i> = 5) or seven (<i>n</i> = 5) days and cryostat brain sections were labeled with TUNEL (red), DAPI (white) and antibodies against GFAP (green). Confocal micrographs show dead cells within the astrocytes at (B–E) one, (F–I) three and (J–M) seven days post-injury. (N) Scatter plot of the percent TUNEL+ cells associated with viable astrocytes in each animal 7 days post-CCI. For each five animals, two separate sections from bregma levels −1.0 and −2.0 mm, were counted and the percentage in 8–10 fields for each level (40× magnification) were plotted. Scale bars = 5 µm.</p

Bystander cell death is induced after direct contact between free-floating cell corpses and healthy, migrating neurons.

<p>(A) The cell corpse (yellow arrowhead) is initially connected to the healthy neuron's (red star) axon (0 h), but apoptosis is not induced until the two cell bodies make contact. After the cell body contacts the cell corpse (1 h 10 min, yellow arrowhead), the healthy neuron (red star) rounds up (1 h 20 min), blebs (1 h 30 min) and dies (2 h). First picture is taken 5 h and 50 minutes after injury and time indicated in the pictures is time lapsed after the first image. Scale bars = 10 µm, the dashed lines represent the scratch. (B) To analyze the frequency of bystander cell death, all neurons in 4 different time-lapse films were tracked. In total we found 26 neurons that came in contact with free-floating dead cells. Of these 26 neurons, 21 neurons (80.8%) died within the experiment. The squares represent the survival time for each of these 21 neurons after contact with the dead cell and lines represent the median±interquartile range.</p

Neurons, astrocytes and oligodendrocytes respond differently to injury <i>in vitro</i>.

<p>(A) Phase contrast micrograph of a scratch injury to a mixed cell culture of neurons, astrocytes and oligodendrocytes. Injury was induced with a scalpel and 22 hours after the cut the cell cultures were fixed in 4% PFA and stained with specific antibodies against (B) neurons (β III tubulin, red), (C) astrocytes (GFAP, green) and (D) oligodendrocytes (CNPase, red). (E–F) Many TUNEL positive cells (green) with condensed nuclei (blue) were found to be in close contact with (E) GFAP positive astrocytes, but not with (F) neurons and only occasionally with (G) oligodendrocytes. Most of the (H) nestin positive cells were associated with TUNEL positive cells. (I) Quantification of dead, TUNEL positive cells that overlapped with either cell type show that astrocytes are the primary cell to be associated with dead cells. Dashed lines represent the scratch. Scale bars equal 50 µm (A–D) and 20 µm (E–H).</p

Astrocytes engulf dead cells following neural injury.

<p>(A–B) Embryonic astrocytes have engulfed dead cells with highly condensed nuclei. Twenty-two hours after scratch injury, the cultures were fixed and astrocytes were identified with specific antibodies against GFAP. Phalloidin and DAPI labeling was used in order to visualize the actin cytoskeleton and cell nuclei, respectively. Condensed DAPI positive nuclei are present in cytoplasmic vacuoles (white arrows) within the viable astrocyte (white asterisk) and not in direct contact with the cytoskeleton of the astrocyte, indicating a macropinocytotic engulfment mechanism. (C) To prove that the cells with condensed nuclei found within astrocytes are <i>de facto</i> dead, we labeled cultures with the apoptotic marker, TUNEL. (D) TEM image of an astrocyte that appear to be in the process of engulfing a dead cell (red star). The dead cell displays the hallmark chromatin traits of apoptosis, but also secondary necrosis as seen by the ruptured cell membrane. The astrocyte contains a previously ingested dead cell (white star) in a spacious vesicle. The astrocyte is marked by an A and the nucleus is denoted Nu. (D') The area marked by a yellow rectangle in D at higher magnification. The ingested dead cell (white star) is contained in a membrane-enclosed compartment (black arrowheads) that is closely linked to the membrane on one side of the dead cell and then diverge to create the vesicle (denoted V) seen in D. Scale bars: 20 µm (A–C), 5 µm (D), 200 nm (D').</p

Inflammatory cells in the injured wt and <i>Ccr2</i>−/− cortex examined by flow cytometry and qRT-PCR.

<p>(<b>a</b>) Cells detected with FITC-CD45 in combination with either APC-PDCA-1 (<i>Bst2</i>) or APC-Cd11c (<i>Itgax</i>) in neocortex of wt or <i>Ccr2</i>−/− mice three days after injury. Upper panel left shows gating for Cd11c. The middle and right panels show wt and <i>Ccr2</i>−/− results, respectively, demonstrating reduced number of Cd11c-positive cells in the <i>Ccr2</i>−/− mice. Lower panel shows that PDCA-1 positive cells were reduced in <i>Ccr2</i>−/− brains. (<b>b</b>) Quantitative flow cytometry data from wt and <i>Ccr2</i>−/− mice three days postinjury. Isotype controls showed only trace signals. (<b>c</b>) Temporal expression patterns of six inflammatory-related transcripts (<i>Itgax</i>, <i>H2-Aa</i>, <i>Bst2</i>, <i>Cxcl10</i>, <i>Lyz2</i> and <i>Hmox1</i>) after injury in wt and <i>Ccr2</i>−/− brains.</p

Chemokine ligands and receptors relevant for this report.

<p>Chemokine ligands and receptors relevant for this report.</p

Study design.

<p>Study design.</p