Perivascular microglia promote blood vessel disintegration in the ischemic penumbra
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The contribution of microglia to ischemic cortical stroke is of particular therapeutic interest because of the impact on the survival of brain tissue in the ischemic penumbra, a region that is potentially salvable upon a brain infarct. Whether or not tissue in the penumbra survives critically depends on blood flow and vessel perfusion. To study the role of microglia in cortical stroke and blood vessel stability, CX3CR1+/GFP mice were subjected to transient middle cerebral artery occlusion and then microglia were investigated using time-lapse two-photon microscopy in vivo. Soon after reperfusion, microglia became activated in the stroke penumbra and started to expand cellular protrusions towards adjacent blood vessels. All microglia in the penumbra were found associated with blood vessels within 24 h post reperfusion and partially fully engulfed them. In the same time frame blood vessels became permissive for blood serum components. Migration assays in vitro showed that blood serum proteins leaking into the tissue provided molecular cues leading to the recruitment of microglia to blood vessels and to their activation. Subsequently, these perivascular microglia started to eat up endothelial cells by phagocytosis, which caused an activation of the local endothelium and contributed to the disintegration of blood vessels with an eventual break down of the blood brain barrier. Loss-of-microglia-function studies using CX3CR1GFP/GFP mice displayed a decrease in stroke size and a reduction in the extravasation of contrast agent into the brain penumbra as measured by MRI. Potentially, medication directed at inhibiting microglia activation within the first day after stroke could stabilize blood vessels in the penumbra, increase blood flow, and serve as a valuable treatment for patients suffering from ischemic stroke.
KeywordsBlood-brain barrier Endothelial cell Ischemic stroke Microglia Middle cerebral artery occlusion
We thank Heike Ehrengard, Christin Liefländer, Christine Oswald, and Andreas Zymny for their excellent technical assistance and Darragh O’Neill for proofreading the manuscript. This work was supported by the Foundation Rhineland-Palatinate to FZ, by the German Research Foundation (DFG) via the collaborative research center 1080, projects A3 (MHHS) and B6 (MKS+FZ), DFG FOR1336 (AW) as well as the DFG Grant SCHM 2159/2-1 to MHHS.
Conflict of interest
The authors declare no competing financial interests.
Suppl. Movie 2 At 8 h after reperfusion perivascular microglia assume a spherical shape in the ischemic penumbra. In this 4.5-h-long time-lapse movie starting 4 h post reperfusion, activation of microglia was measured by intravital two-photon microscopy. At 8 h post reperfusion, microglia cell bodies changed from an elongated to a spherical shape (arrows). GFP-positive microglia are indicated in green, rhodamine–dextran-labeled blood vessels in red. Each frame in this movie represents a z-projection of a 70-µm-thick stack acquired at 1-min intervals in vivo (AVI 13793 kb)
Suppl. Movie 3 At 8 h after reperfusion microglia migrate towards blood vessels. In this 55-min-long time-lapse movie starting 8 h post reperfusion, intravital two-photon microscopy revealed the migration of GFP-positive microglia (green) towards rhodamine–dextran-labeled blood vessels (red) 8 h post reperfusion (arrows). Each frame in this movie represents a z-projection of a 70-µm-thick stack acquired at 1-min intervals in vivo (AVI 2064 kb)
Suppl. Movie 4 At 8 h after reperfusion a microglia cell migrates towards a blood vessel. This movie shows a magnification of a cell derived from supplementary movie 3 migrating towards a blood vessel 8 h post reperfusion. Each frame in this movie represents a z-projection of a 70-µm-thick stack acquired at 1-min intervals in vivo (AVI 300 kb)
Suppl. Movie 5 At 24 h post reperfusion microglia phagocytize blood vessel components. This 70-min-long intravital two-photon microscopy time-lapse movie shows GFP-positive microglia (green) aligning with rhodamine–dextran-labeled blood vessels (red, small arrow). At 24 h post reperfusion these cells started to phagocytize rhodamine–dextran-positive blood vessel components (yellow, big arrow). Each frame in this movie represents a z-projection of a 70-µm-thick stack acquired at 1-min intervals in vivo (AVI 4281 kb)
Suppl. Movie 6 At 24 h post reperfusion microglia disintegrate blood vessels. This 88-min-long intravital two-photon microscopy time-lapse movie shows GFP-positive activated microglia (green) that are mainly associated with blood vessels 24 h post reperfusion. Two of the microglia cells actively migrate towards blood vessels (big arrows) and scan them with their processes. The disintegration of the blood vasculature has already started, as demonstrated by patchy extravasation of rhodamine–dextran in the lower part of the video (small arrows). Each frame in this movie represents a z-projection of a 70-µm-thick stack acquired at 1-min intervals in vivo (AVI 7722 kb)
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