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Very-late-antigen-4 (VLA-4)-mediated brain invasion by neutrophils leads to interactions with microglia, increased ischemic injury and impaired behavior in experimental stroke

Abstract

Neuronal injury from ischemic stroke is aggravated by invading peripheral immune cells. Early infiltrates of neutrophil granulocytes and T-cells influence the outcome of stroke. So far, however, neither the timing nor the cellular dynamics of neutrophil entry, its consequences for the invaded brain area, or the relative importance of T-cells has been extensively studied in an intravital setting. Here, we have used intravital two-photon microscopy to document neutrophils and brain-resident microglia in mice after induction of experimental stroke. We demonstrated that neutrophils immediately rolled, firmly adhered, and transmigrated at sites of endothelial activation in stroke-affected brain areas. The ensuing neutrophil invasion was associated with local blood–brain barrier breakdown and infarct formation. Brain-resident microglia recognized both endothelial damage and neutrophil invasion. In a cooperative manner, they formed cytoplasmic processes to physically shield activated endothelia and trap infiltrating neutrophils. Interestingly, the systemic blockade of very-late-antigen-4 immediately and very effectively inhibited the endothelial interaction and brain entry of neutrophils. This treatment thereby strongly reduced the ischemic tissue injury and effectively protected the mice from stroke-associated behavioral impairment. Behavioral preservation was also equally well achieved with the antibody-mediated depletion of myeloid cells or specifically neutrophils. In contrast, T-cell depletion more effectively reduced the infarct volume without improving the behavioral performance. Thus, neutrophil invasion of the ischemic brain is rapid, massive, and a key mediator of functional impairment, while peripheral T-cells promote brain damage. Acutely depleting T-cells and inhibiting brain infiltration of neutrophils might, therefore, be a powerful early stroke treatment.

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Acknowledgments

We thank Susanne v. Kenne for excellent technical assistance. For help with flow cytometry, we thank Stefanie Holze and Jenny Schneeberg, and the Imaging Center Essen (IMCES) for help with imaging. This work was supported by the German Research foundation (DFG, SFB 854 to M.G. and K.R. as well as SPP1468 “Immunobone” to M.G. and HE3173/2-1 and HE3173/3-1 to D.M.H.), a DZNE intersite project on vascular dementia to K.R. and the Mercator Research Center Ruhr (An-2011-0081 to JH).

Author information

Correspondence to Matthias Gunzer.

Additional information

J. Neumann, M. Riek-Burchardt, J. Herz, D. M. Hermann, K. G. Reymann and M. Gunzer contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 8 (AVI 195 kb) Supplemental Movie 1 Microglial morphology and motility in response to cerebral ischemia. The movie shows two-photon intravital microscopy of microglia (CX3CR-1-eGFP mice) distant (left side) and more closely positioned to the ischemic core (right side) 24h after pMCAO. Please note the profound membrane motility of the cells close to the ischemic core as opposed to cells more distant that are relatively quiet. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 9 (MP4 181 kb) Supplemental Movie 1 Microglial morphology and motility in response to cerebral ischemia. The movie shows two-photon intravital microscopy of microglia (CX3CR-1-eGFP mice) distant (left side) and more closely positioned to the ischemic core (right side) 24h after pMCAO. Please note the profound membrane motility of the cells close to the ischemic core as opposed to cells more distant that are relatively quiet. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 12 (AVI 1282 kb) Supplemental Movie 3 The movie illustrates a slightly enhanced rolling of PMN visualized by two-photon intravital microscopy in Lys-eGFP mice in an area not in the immediate vicinity to the ischemic core (corresponding to Fig. 1a; Area 2). Real time of the experiment is shown inminutes. The scale bar is 50 μm

Supplementary material 13 (MP4 1251 kb) Supplemental Movie 3 The movie illustrates a slightly enhanced rolling of PMN visualized by two-photon intravital microscopy in Lys-eGFP mice in an area not in the immediate vicinity to the ischemic core (corresponding to Fig. 1a; Area 2). Real time of the experiment is shown inminutes. The scale bar is 50 μm

Supplementary material 24 (AVI 11438 kb) Supplemental Movie 9 Adhering and extravasation of PMN after thrombus formation induced by severe laser irradiation. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24h after ischemia (corresponding to area 1 in Fig. 1a). The movie shows rolling and transiently adhering PMN before laser irradiation and thereafter at the beginning the thrombus formation, followed by a massive accumulation of PMN, the physical breakdown of the blood brain barrier (around 53 min) and finally a tremendous extravasation of PMN. Real time of the experiment is shown in minutes. The scale bar is 30 µm

Supplementary material 25 (MP4 11418 kb) Supplemental Movie 9 Adhering and extravasation of PMN after thrombus formation induced by severe laser irradiation. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24h after ischemia (corresponding to area 1 in Fig. 1a). The movie shows rolling and transiently adhering PMN before laser irradiation and thereafter at the beginning the thrombus formation, followed by a massive accumulation of PMN, the physical breakdown of the blood brain barrier (around 53 min) and finally a tremendous extravasation of PMN. Real time of the experiment is shown in minutes. The scale bar is 30 µm

Supplementary material 36 (AVI 5632 kb) Supplemental Movie 15 Microglial morphology of CX3CR1-eGFP cells is associated with a Z-level below the focal plane of the meninges. A 2-Photon microscopy in-situ Z-stack of a CX3CR1-eGFP brain was reconstructed with 3D animation via IMARIS. The meninges were identified by their SHG signal (grey) and embedded blood vessels by PECAM1 immunostaining (red). The brain itself shows no SHG signal, but bright staining of capillaries (red). CX3CR1-GFP positive microglia (green) are found in the whole brain starting beneath the level of the meninges, whereas macrophages/monocytes (green round cells) are exclusively found attached to the meninges. Immunofluorescence staining of blood vessels: 10µg/mouse PE-anti-PECAM1 (BD, Cat: 553373) were intravenously injected. 20min after injection the mouse was perfused with PBS to remove unbound antibodies. The skull was opened and the brain was directly imaged in 37°C warmed PBS. 2-Photon setup: for imaging the CX3CR1-GFP mouse brain the 2-Photon laser was tuned to 960nm for GFP. For the ensuing simultaneous detection of PE and SHG an OPO was used at 1.100 nm excitation with SHG detection at 550nm

Supplementary material 37 (MP4 7867 kb) Supplemental Movie 15 Microglial morphology of CX3CR1-eGFP cells is associated with a Z-level below the focal plane of the meninges. A 2-Photon microscopy in-situ Z-stack of a CX3CR1-eGFP brain was reconstructed with 3D animation via IMARIS. The meninges were identified by their SHG signal (grey) and embedded blood vessels by PECAM1 immunostaining (red). The brain itself shows no SHG signal, but bright staining of capillaries (red). CX3CR1-GFP positive microglia (green) are found in the whole brain starting beneath the level of the meninges, whereas macrophages/monocytes (green round cells) are exclusively found attached to the meninges. Immunofluorescence staining of blood vessels: 10µg/mouse PE-anti-PECAM1 (BD, Cat: 553373) were intravenously injected. 20min after injection the mouse was perfused with PBS to remove unbound antibodies. The skull was opened and the brain was directly imaged in 37°C warmed PBS. 2-Photon setup: for imaging the CX3CR1-GFP mouse brain the 2-Photon laser was tuned to 960nm for GFP. For the ensuing simultaneous detection of PE and SHG an OPO was used at 1.100 nm excitation with SHG detection at 550nm

Supplementary material 1 (TIFF 2573 kb) Supplemental Fig. 1.Leakage of rhodamine dextran from the blood vessel after laser damage. The first two images (from left) show a small bleeding (arrows) after irritation of the bloodvessel by enhanced laser exposure. The last two images demonstrate a spontaneous bleeding (no enhanced, focussed laser beam at this vessel part) due to a much higher laser exposure than normally applied. (Supplemental movie 7) Scale bar: 20 µm

Supplementary material 2 (TIFF 1281 kb) Supplemental Fig. 2 Lack of recruitment of PMN in mice without experimental ischemic injury. The first image represents the basal condition of blood flow in the brain of healthy Lys-eGFP mice before vessel irritation by enhanced laser exposure. The flash shows the localisation of the irritation. The second image shows no recruitment of eGFPbright cells towards the endothelium. To induce further damage, the same location was irritated by laser again. This led to a coagulation of the vessel, which is displayed by arrows in the third image. Overall a recruitment of eGFPbright cells was not observed during the whole procedure (Supplemental movie 11). Scale bar: 20 µm

Supplementary material 3 (TIFF 974 kb) Supplemental Fig. 3 FACS analysis of the expression of VLA-4 on PMN in the blood of a human donor. The data are representative of 2 healthy volunteers as well as 2 patients (24 h after onset of stroke) without showing differences in the VLA-4 expression between both groups

Supplementary material 4 (TIFF 584 kb) Supplemental Fig. 4 FACS analysis of the expression of GFP in PMN and macrophages of Lys-eGFP mice. Blood of Lys-eGFP mice was labeled with anti-Ly6G PE and anti-Gr-1 APC. Leukocytes were gated by forward/sideward scatter and are shown in the 1A8-PE and Gr-1-APC Plot. The 1A8bright/Gr-1high population (neutrophils, gate 1) also expresses eGFP at a very high level (histogram on the right). All Gr-1medium/1A8negative cells (monocytes/macrophages, gate 2) express ~1 log less eGFP (histogram on the right). Gr-1negative cells (other myeloid cells, lymphocytes) are also negative for eGFP (not shown)

Supplementary material 5 (TIFF 1507 kb) Supplemental Fig. 5 Analysis of the migration speed of PMN in the brain of mice. PMN migration within the penumbra 24h after pMCAO was recorded by in vivo 2-photon microscopy in Lys-eGFP mice in a depth of 100µm below the meninges. Movies were analyzed by semi-automated single cell tracking using IMARIS. PMN display a mean speed of 10 ± 6.1 µm per min. Data are shown as mean values ± SD from 3 independent experiments

Supplementary material 6 (TIFF 473 kb) Supplemental Fig. 6 Analysis of the distance of brain-resident PMN from blood vessels. a The distance between PMN and nearby vessels was measured using Z-stacks from in vivo recordings by 2-photon microscopy in Lys-eGFP mice 24h after pMCAO in 3 independent experiments. We found different PMN populations which are either next to the endothelium (0µm), less than 10µm away (mean 7.2± 3 µm) or more than 10µm away (mean 18.2 ± 4.6 µm) from the nearest blood vessel. b Representative images of a brain section stained for Ly6G (green), CD31 (red) and nuclei (blue) showing infiltrated and vessel-associated PMN in ischemic brain tissue 72h post 45 minutes tMCAO. c the distance between neutrophils and anti-CD31-stained vessels was measured in confocal images (6-10 ROI/animal) of stainings shown in b. A total of 251 cells out of 4 animals was analysed. d single values of distances for cells > 10 µm are shown and summarized as mean ± SD. Scale bar: 50 µm. ***p < 0.001

Supplementary material 7 (TIFF 3502 kb) Supplemental Fig. 7 Analysis of edema formation after immune cell subset depletion combined with anti-VLA-4 treatment after 45 minutes tMCAO. The ratio of ipsilateral hemisphere volumes to contralateral volumes was calculated as a measure of edema formation for the indicated experimental groups. Treatment paradigms are given in Fig. 7a and 8d. Data are mean + SD (9-12 animals per group). *p < 0.05

Supplementary material 8 (AVI 195 kb) Supplemental Movie 1 Microglial morphology and motility in response to cerebral ischemia. The movie shows two-photon intravital microscopy of microglia (CX3CR-1-eGFP mice) distant (left side) and more closely positioned to the ischemic core (right side) 24h after pMCAO. Please note the profound membrane motility of the cells close to the ischemic core as opposed to cells more distant that are relatively quiet. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 9 (MP4 181 kb) Supplemental Movie 1 Microglial morphology and motility in response to cerebral ischemia. The movie shows two-photon intravital microscopy of microglia (CX3CR-1-eGFP mice) distant (left side) and more closely positioned to the ischemic core (right side) 24h after pMCAO. Please note the profound membrane motility of the cells close to the ischemic core as opposed to cells more distant that are relatively quiet. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 10 (AVI 1730 kb) Supplemental Movie 2 Rolling, adhering and extravasation of PMN after cerebral ischemia. The movie shows two-photon intravital microscopy of a bloodvessel in the vicinity of the ischemic core (corresponding to Fig. 1a; Area 1) in Lys-eGFP mice. The tracks indicate extravasation of three PMN into the brain parenchyma. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 11 (MP4 1695 kb) Supplemental Movie 2 Rolling, adhering and extravasation of PMN after cerebral ischemia. The movie shows two-photon intravital microscopy of a bloodvessel in the vicinity of the ischemic core (corresponding to Fig. 1a; Area 1) in Lys-eGFP mice. The tracks indicate extravasation of three PMN into the brain parenchyma. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 12 (AVI 1282 kb) Supplemental Movie 3 The movie illustrates a slightly enhanced rolling of PMN visualized by two-photon intravital microscopy in Lys-eGFP mice in an area not in the immediate vicinity to the ischemic core (corresponding to Fig. 1a; Area 2). Real time of the experiment is shown inminutes. The scale bar is 50 μm

Supplementary material 13 (MP4 1251 kb) Supplemental Movie 3 The movie illustrates a slightly enhanced rolling of PMN visualized by two-photon intravital microscopy in Lys-eGFP mice in an area not in the immediate vicinity to the ischemic core (corresponding to Fig. 1a; Area 2). Real time of the experiment is shown inminutes. The scale bar is 50 μm

Supplementary material 14 (AVI 524 kb) Supplemental Movie 4 Morphology of microglia and endothelial interaction of PMN beyond the infarcted area. The movie shows two-photon intravital microscopy in double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) (Fig. 1a; Area 3) normal microglial morphology and no enhanced interaction of PMN (PMN only detectable as green stripes due to their high velocity freely within blood vessels) with the endothelium. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 15 (MP4 480 kb) Supplemental Movie 4 Morphology of microglia and endothelial interaction of PMN beyond the infarcted area. The movie shows two-photon intravital microscopy in double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) (Fig. 1a; Area 3) normal microglial morphology and no enhanced interaction of PMN (PMN only detectable as green stripes due to their high velocity freely within blood vessels) with the endothelium. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 16 (AVI 416 kb) Supplemental Movie 5 Absence of green cells interacting with the inflamed endothelium after stroke in CX3CR1-eGFP mice. Two- photon intravital microscopy was performed within CX3CR1-eGFP transgenic mice 24h after ischemia. In the movie the blue track shows the migration path of only one detected eGFP+ cell rolling on the inflamed endothelium of the infarcted brain. In contrast numerous other cells roll and adhere on the inflamed endothelium (black shadows within the vessel) which do not express eGFP and therefore do not represent CX3CR-1+ macrophages. Real time of the experiment is shown in minutes. The scale bar is 30 µm

Supplementary material 17 (MP4 405 kb) Supplemental Movie 5 Absence of green cells interacting with the inflamed endothelium after stroke in CX3CR1-eGFP mice. Two- photon intravital microscopy was performed within CX3CR1-eGFP transgenic mice 24h after ischemia. In the movie the blue track shows the migration path of only one detected eGFP+ cell rolling on the inflamed endothelium of the infarcted brain. In contrast numerous other cells roll and adhere on the inflamed endothelium (black shadows within the vessel) which do not express eGFP and therefore do not represent CX3CR-1+ macrophages. Real time of the experiment is shown in minutes. The scale bar is 30 µm

Supplementary material 18 (AVI 948 kb) Supplemental Movie 6 Microglia cooperate to trap invading PMN. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24h after ischemia. The movie illustrates the migration of PMN. After contact microglia send multiple membrane protrusions towards PMN and cooperate with other microglia to form net-like structures (white square). Please note, that many PMN can be seen entering a net, but very few appear to leave it again. The tracks show the migration path of PMN to the site of trapping. Real time of the experiment is shown in minutes. The scale bar is 10 µm

Supplementary material 19 (MP4 939 kb) Supplemental Movie 6 Microglia cooperate to trap invading PMN. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24h after ischemia. The movie illustrates the migration of PMN. After contact microglia send multiple membrane protrusions towards PMN and cooperate with other microglia to form net-like structures (white square). Please note, that many PMN can be seen entering a net, but very few appear to leave it again. The tracks show the migration path of PMN to the site of trapping. Real time of the experiment is shown in minutes. The scale bar is 10 µm

Supplementary material 20 (AVI 442 kb) Supplemental Movie 7 Blood vessel leakage after heavy laser damage. Two-photon intravital microscopy in CX3CR1-eGFP transgenic mice shows bleeding after an irritation of the blood vessel by enhanced laser exposure and thereafter a spontaneous intense bleeding due to a repetitive exposure to a much higher laser exposure than normally applied. After the vessel disruption the rhodamine-dextrane immediately leaks into the surrounding tissue. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 21 (MP4 434 kb) Supplemental Movie 7 Blood vessel leakage after heavy laser damage. Two-photon intravital microscopy in CX3CR1-eGFP transgenic mice shows bleeding after an irritation of the blood vessel by enhanced laser exposure and thereafter a spontaneous intense bleeding due to a repetitive exposure to a much higher laser exposure than normally applied. After the vessel disruption the rhodamine-dextrane immediately leaks into the surrounding tissue. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 22 (AVI 2542 kb) Supplemental Movie 8 PMN and microglia sense endothelial activation after mild laser irradiation. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24h after ischemia in area 2 (Fig. 1a). The movie shows the free flowing and slight endothelial interaction of PMN before and the immediate accumulation of PMN and the reaction of microglia (membrane protrusions) after short laser irradiation. Please note the specific localization of PMN only at the site of laser irradiation (arrow). Real time of the experiment is shown in minutes. The scale bar is 40 µm

Supplementary material 23 (MP4 2545 kb) Supplemental Movie 8 PMN and microglia sense endothelial activation after mild laser irradiation. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24h after ischemia in area 2 (Fig. 1a). The movie shows the free flowing and slight endothelial interaction of PMN before and the immediate accumulation of PMN and the reaction of microglia (membrane protrusions) after short laser irradiation. Please note the specific localization of PMN only at the site of laser irradiation (arrow). Real time of the experiment is shown in minutes. The scale bar is 40 µm

Supplementary material 24 (AVI 11438 kb) Supplemental Movie 9 Adhering and extravasation of PMN after thrombus formation induced by severe laser irradiation. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24h after ischemia (corresponding to area 1 in Fig. 1a). The movie shows rolling and transiently adhering PMN before laser irradiation and thereafter at the beginning the thrombus formation, followed by a massive accumulation of PMN, the physical breakdown of the blood brain barrier (around 53 min) and finally a tremendous extravasation of PMN. Real time of the experiment is shown in minutes. The scale bar is 30 µm

Supplementary material 25 (MP4 11418 kb) Supplemental Movie 9 Adhering and extravasation of PMN after thrombus formation induced by severe laser irradiation. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24h after ischemia (corresponding to area 1 in Fig. 1a). The movie shows rolling and transiently adhering PMN before laser irradiation and thereafter at the beginning the thrombus formation, followed by a massive accumulation of PMN, the physical breakdown of the blood brain barrier (around 53 min) and finally a tremendous extravasation of PMN. Real time of the experiment is shown in minutes. The scale bar is 30 µm

Supplementary material 26 (AVI 5786 kb) Supplemental Movie 10 Rapid recruitment of PMN to the endothelium after thrombus formation and crawling against the blood flow. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24 h after ischemia (in a zone corresponding to area 2 in Fig. 1a). The movie illustrates the free flowing of PMN before and the inflammatory response after thrombus formation. Please note the rapid recruitment of PMN to a vessel segment which is located beneath the thrombus and PMN which crawl against the bloodstream to the lesioned site. The tracks show the migration path along the vessel wall to the thrombus. Please also note the profound morphological changes of most microglia visible within the brain parenchyma after onset of thrombus formation. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 27 (MP4 5886 kb) Supplemental Movie 10 Rapid recruitment of PMN to the endothelium after thrombus formation and crawling against the blood flow. Two-photon intravital microscopy was performed within double transgenic mice (Lys-M-eGFP/ CX3CR-1-eGFP) 24 h after ischemia (in a zone corresponding to area 2 in Fig. 1a). The movie illustrates the free flowing of PMN before and the inflammatory response after thrombus formation. Please note the rapid recruitment of PMN to a vessel segment which is located beneath the thrombus and PMN which crawl against the bloodstream to the lesioned site. The tracks show the migration path along the vessel wall to the thrombus. Please also note the profound morphological changes of most microglia visible within the brain parenchyma after onset of thrombus formation. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 28 (AVI 1833 kb) Supplemental Movie 11 No recruitment of PMN in the absence of stroke. Two- photon intravital microscopy was performed in Lys-eGFP transgenic mice without previous ischemia. The first part of the movie represents the basal condition in Lys-eGFP mice before vessel irritation by enhanced laser exposure. The white arrow displays the site of enhanced laser exposure in part two of the movie. In part three, the same location was irritated by laser again to induce further damage which finally led to a coagulation of the vessel (black arrow). Overall a recruitment of PMN was not observed during the whole procedure. Real time of the experiment is shown in minutes. The scale bar is 20µm

Supplementary material 29 (MP4 1823 kb) Supplemental Movie 11 No recruitment of PMN in the absence of stroke. Two- photon intravital microscopy was performed in Lys-eGFP transgenic mice without previous ischemia. The first part of the movie represents the basal condition in Lys-eGFP mice before vessel irritation by enhanced laser exposure. The white arrow displays the site of enhanced laser exposure in part two of the movie. In part three, the same location was irritated by laser again to induce further damage which finally led to a coagulation of the vessel (black arrow). Overall a recruitment of PMN was not observed during the whole procedure. Real time of the experiment is shown in minutes. The scale bar is 20µm

Supplementary material 30 (AVI 1665 kb) Supplemental Movie 12 Impact of anti-VLA-4 treatment on endothelial interaction of PMN. Two-photon intravital microscopy was performed in Lys-eGFP-mice 24h after ischemia. The movie shows on the left side the adherence and rolling of PMN in the vicinity to the ischemic core (corresponding to area 1 in Fig. 1a). The right side illustrates the PMN-endothelium interaction in the same area 15 minutes after anti-VLA-4 treatment. Real time of the experiment is shown in minutes. The scale bar is 30 µm

Supplementary material 31 (MP4 1611 kb) Supplemental Movie 12 Impact of anti-VLA-4 treatment on endothelial interaction of PMN. Two-photon intravital microscopy was performed in Lys-eGFP-mice 24h after ischemia. The movie shows on the left side the adherence and rolling of PMN in the vicinity to the ischemic core (corresponding to area 1 in Fig. 1a). The right side illustrates the PMN-endothelium interaction in the same area 15 minutes after anti-VLA-4 treatment. Real time of the experiment is shown in minutes. The scale bar is 30 µm

Supplementary material 32 (AVI 397 kb) Supplemental Movie 13 PMN interact with the inflamed endothelium and invade the parenchyma of the brain after isotype treatment and pMCAO. Two-photon intravital microscopy was performed in Lys-eGFP transgenic mice 24h after pMCAO. Before induction of cerebral ischemia mice had been treated with isotype antibody. The movie shows a number of PMN (eGFP+ cells) interacting with the inflamed endothelium (adherence and rolling) as well as PMN migrating within the parenchyma and in the tissue around the blood vessels. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 33 (MP4 377 kb) Supplemental Movie 13 PMN interact with the inflamed endothelium and invade the parenchyma of the brain after isotype treatment and pMCAO. Two-photon intravital microscopy was performed in Lys-eGFP transgenic mice 24h after pMCAO. Before induction of cerebral ischemia mice had been treated with isotype antibody. The movie shows a number of PMN (eGFP+ cells) interacting with the inflamed endothelium (adherence and rolling) as well as PMN migrating within the parenchyma and in the tissue around the blood vessels. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 34 (AVI 1421 kb) Supplemental Movie 14 Lack of PMN interacting with the inflamed endothelium and invading the brain after 1A8 treatment and pMCAO. Two-photon intravital microscopy was performed in Lys-eGFP transgenic mice 24h after pMCAO. Before induction of cerebral ischemia mice had been treated with depleting doses of 1A8 antibody. The movie illustrates very few PMN (eGFP+ cells) interacting with inflamed endothelium or migrating in the parenchyma and in the tissue around the blood vessels. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 35 (MP4 1393 kb) Supplemental Movie 14 Lack of PMN interacting with the inflamed endothelium and invading the brain after 1A8 treatment and pMCAO. Two-photon intravital microscopy was performed in Lys-eGFP transgenic mice 24h after pMCAO. Before induction of cerebral ischemia mice had been treated with depleting doses of 1A8 antibody. The movie illustrates very few PMN (eGFP+ cells) interacting with inflamed endothelium or migrating in the parenchyma and in the tissue around the blood vessels. Real time of the experiment is shown in minutes. The scale bar is 20 µm

Supplementary material 36 (AVI 5632 kb) Supplemental Movie 15 Microglial morphology of CX3CR1-eGFP cells is associated with a Z-level below the focal plane of the meninges. A 2-Photon microscopy in-situ Z-stack of a CX3CR1-eGFP brain was reconstructed with 3D animation via IMARIS. The meninges were identified by their SHG signal (grey) and embedded blood vessels by PECAM1 immunostaining (red). The brain itself shows no SHG signal, but bright staining of capillaries (red). CX3CR1-GFP positive microglia (green) are found in the whole brain starting beneath the level of the meninges, whereas macrophages/monocytes (green round cells) are exclusively found attached to the meninges. Immunofluorescence staining of blood vessels: 10µg/mouse PE-anti-PECAM1 (BD, Cat: 553373) were intravenously injected. 20min after injection the mouse was perfused with PBS to remove unbound antibodies. The skull was opened and the brain was directly imaged in 37°C warmed PBS. 2-Photon setup: for imaging the CX3CR1-GFP mouse brain the 2-Photon laser was tuned to 960nm for GFP. For the ensuing simultaneous detection of PE and SHG an OPO was used at 1.100 nm excitation with SHG detection at 550nm

Supplementary material 37 (MP4 7867 kb) Supplemental Movie 15 Microglial morphology of CX3CR1-eGFP cells is associated with a Z-level below the focal plane of the meninges. A 2-Photon microscopy in-situ Z-stack of a CX3CR1-eGFP brain was reconstructed with 3D animation via IMARIS. The meninges were identified by their SHG signal (grey) and embedded blood vessels by PECAM1 immunostaining (red). The brain itself shows no SHG signal, but bright staining of capillaries (red). CX3CR1-GFP positive microglia (green) are found in the whole brain starting beneath the level of the meninges, whereas macrophages/monocytes (green round cells) are exclusively found attached to the meninges. Immunofluorescence staining of blood vessels: 10µg/mouse PE-anti-PECAM1 (BD, Cat: 553373) were intravenously injected. 20min after injection the mouse was perfused with PBS to remove unbound antibodies. The skull was opened and the brain was directly imaged in 37°C warmed PBS. 2-Photon setup: for imaging the CX3CR1-GFP mouse brain the 2-Photon laser was tuned to 960nm for GFP. For the ensuing simultaneous detection of PE and SHG an OPO was used at 1.100 nm excitation with SHG detection at 550nm

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Neumann, J., Riek-Burchardt, M., Herz, J. et al. Very-late-antigen-4 (VLA-4)-mediated brain invasion by neutrophils leads to interactions with microglia, increased ischemic injury and impaired behavior in experimental stroke. Acta Neuropathol 129, 259–277 (2015) doi:10.1007/s00401-014-1355-2

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Keywords

  • Stroke
  • Intravital 2-photon microscopy
  • Neutrophils
  • VLA-4