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In Vivo Non-Invasive Tracking of Macrophage Recruitment to Experimental Stroke.

Selt M, Tennstaedt A, Beyrau A, Nelles M, Schneider G, Löwik C, Hoehn M - PLoS ONE (2016)

Bottom Line: However, the underlying molecular mechanisms needed for proper regulation still remain to be elucidated.The labelling procedure of the isolated cells did not significantly influence cell characteristics and resulted in detection of as few as 500 labelled cells in vivo.Hypointensity in MRI of the graft appeared unaltered in spatial location.

View Article: PubMed Central - PubMed

Affiliation: In-vivo-NMR Laboratory, Max Planck Institute for Metabolism Research, Cologne, Germany.

ABSTRACT
Brain-infiltrating monocyte-derived macrophages are one of the key players in the local immune response after stroke. It is now widely accepted that the inflammatory response is not an exclusively destructive process. However, the underlying molecular mechanisms needed for proper regulation still remain to be elucidated. Here, we propose an in vitro labelling strategy for multimodal in vivo observation of macrophage dynamics distinguished from brain-residing microglia response. Prior to intracerebral transplantation into the striatum of recipient mice or systemic administration, monocytes and macrophages, isolated from luciferase-expressing mice, were labelled with superparamagnetic iron oxide particles. Temporo-spatial localization was monitored by magnetic resonance imaging, whereas survival of grafted cells was investigated using bioluminescence imaging. The labelling procedure of the isolated cells did not significantly influence cell characteristics and resulted in detection of as few as 500 labelled cells in vivo. Two weeks after stereotactic transplantation, the luciferase signal was sustained traceable, with approximately 18% of the original luciferase signal detectable for monocytes and about 30% for macrophages. Hypointensity in MRI of the graft appeared unaltered in spatial location. In a therapeutically relevant approach, systemic cell administration after stroke resulted in accumulation mostly in thoracic regions, as could be visualized with BLI. For detection of homing to ischemic brain tissue more cells need to be administered. Nevertheless, during parallel MRI sessions recruitment of i.v. injected cells to the lesion site could be detected by day 2 post stroke as scattered hypointense signal voids. With further increase in sensitivity, our multi-facetted labelling strategy will provide the basis for in vivo tracking and fate specification of tissue-infiltrating macrophages and their distinct role in stroke-related neuro-inflammation.

No MeSH data available.


Related in: MedlinePlus

In vitro iron labelling efficiency of monocytes and MΦ (WT and luc+).(A) Overnight incubation with 168 μg Fe/ml resulted in blue deposits in phase contrast and brightfield microscopy images of PB staining in monocytes and MΦ (scale = 50 μm). (B) Photometric iron content analysis for control (light grey) and contrast agent labelled WT and luc+ cells (dark grey; 168 μg Fe/ml), with 2–6 experiments per cell type and Nanomag concentration. (C) Photometric iron uptake titration of luc+ monocytes and MΦ. Mean photometric values are displayed as pg Fe/cell. Statistical significance (ANOVA/Kruskal-Wallis: * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001)always indicates the difference in iron uptake between luc+ monocytes and luc+ macrophages. Luc+ monocytes: 4–11 experiments per concentration, luc+ Mɸ: 5–7 experiments per concentration.
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pone.0156626.g001: In vitro iron labelling efficiency of monocytes and MΦ (WT and luc+).(A) Overnight incubation with 168 μg Fe/ml resulted in blue deposits in phase contrast and brightfield microscopy images of PB staining in monocytes and MΦ (scale = 50 μm). (B) Photometric iron content analysis for control (light grey) and contrast agent labelled WT and luc+ cells (dark grey; 168 μg Fe/ml), with 2–6 experiments per cell type and Nanomag concentration. (C) Photometric iron uptake titration of luc+ monocytes and MΦ. Mean photometric values are displayed as pg Fe/cell. Statistical significance (ANOVA/Kruskal-Wallis: * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001)always indicates the difference in iron uptake between luc+ monocytes and luc+ macrophages. Luc+ monocytes: 4–11 experiments per concentration, luc+ Mɸ: 5–7 experiments per concentration.

Mentions: Isolated monocytes and differentiated MΦ of C57BL/6 WT and of transgenic β–actin luc mice, as well as MΦ of an immortalized cell line J774A.1, were incubated overnight with Nanomag SPIO particles at a concentration of 168 μg Fe/ml, without the addition of lipofectant agents. Intracellular iron uptake by the cells was clearly visualized by distinct blue deposits after PB staining in phase contrast as well as brightfield microscopy (Fig 1A; S1A Fig), whereas unlabelled control cells remained unstained. Also, ICC staining of control and labelled WT monocytes against the dextran surface coating of the iron oxide particles resulted in clear visualization of the contrast agent particles within the labelled cells (S2 Fig). In contrast, unlabelled control cells did not show fluorescence signal from anti-dextran antibody.


In Vivo Non-Invasive Tracking of Macrophage Recruitment to Experimental Stroke.

Selt M, Tennstaedt A, Beyrau A, Nelles M, Schneider G, Löwik C, Hoehn M - PLoS ONE (2016)

In vitro iron labelling efficiency of monocytes and MΦ (WT and luc+).(A) Overnight incubation with 168 μg Fe/ml resulted in blue deposits in phase contrast and brightfield microscopy images of PB staining in monocytes and MΦ (scale = 50 μm). (B) Photometric iron content analysis for control (light grey) and contrast agent labelled WT and luc+ cells (dark grey; 168 μg Fe/ml), with 2–6 experiments per cell type and Nanomag concentration. (C) Photometric iron uptake titration of luc+ monocytes and MΦ. Mean photometric values are displayed as pg Fe/cell. Statistical significance (ANOVA/Kruskal-Wallis: * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001)always indicates the difference in iron uptake between luc+ monocytes and luc+ macrophages. Luc+ monocytes: 4–11 experiments per concentration, luc+ Mɸ: 5–7 experiments per concentration.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920382&req=5

pone.0156626.g001: In vitro iron labelling efficiency of monocytes and MΦ (WT and luc+).(A) Overnight incubation with 168 μg Fe/ml resulted in blue deposits in phase contrast and brightfield microscopy images of PB staining in monocytes and MΦ (scale = 50 μm). (B) Photometric iron content analysis for control (light grey) and contrast agent labelled WT and luc+ cells (dark grey; 168 μg Fe/ml), with 2–6 experiments per cell type and Nanomag concentration. (C) Photometric iron uptake titration of luc+ monocytes and MΦ. Mean photometric values are displayed as pg Fe/cell. Statistical significance (ANOVA/Kruskal-Wallis: * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001)always indicates the difference in iron uptake between luc+ monocytes and luc+ macrophages. Luc+ monocytes: 4–11 experiments per concentration, luc+ Mɸ: 5–7 experiments per concentration.
Mentions: Isolated monocytes and differentiated MΦ of C57BL/6 WT and of transgenic β–actin luc mice, as well as MΦ of an immortalized cell line J774A.1, were incubated overnight with Nanomag SPIO particles at a concentration of 168 μg Fe/ml, without the addition of lipofectant agents. Intracellular iron uptake by the cells was clearly visualized by distinct blue deposits after PB staining in phase contrast as well as brightfield microscopy (Fig 1A; S1A Fig), whereas unlabelled control cells remained unstained. Also, ICC staining of control and labelled WT monocytes against the dextran surface coating of the iron oxide particles resulted in clear visualization of the contrast agent particles within the labelled cells (S2 Fig). In contrast, unlabelled control cells did not show fluorescence signal from anti-dextran antibody.

Bottom Line: However, the underlying molecular mechanisms needed for proper regulation still remain to be elucidated.The labelling procedure of the isolated cells did not significantly influence cell characteristics and resulted in detection of as few as 500 labelled cells in vivo.Hypointensity in MRI of the graft appeared unaltered in spatial location.

View Article: PubMed Central - PubMed

Affiliation: In-vivo-NMR Laboratory, Max Planck Institute for Metabolism Research, Cologne, Germany.

ABSTRACT
Brain-infiltrating monocyte-derived macrophages are one of the key players in the local immune response after stroke. It is now widely accepted that the inflammatory response is not an exclusively destructive process. However, the underlying molecular mechanisms needed for proper regulation still remain to be elucidated. Here, we propose an in vitro labelling strategy for multimodal in vivo observation of macrophage dynamics distinguished from brain-residing microglia response. Prior to intracerebral transplantation into the striatum of recipient mice or systemic administration, monocytes and macrophages, isolated from luciferase-expressing mice, were labelled with superparamagnetic iron oxide particles. Temporo-spatial localization was monitored by magnetic resonance imaging, whereas survival of grafted cells was investigated using bioluminescence imaging. The labelling procedure of the isolated cells did not significantly influence cell characteristics and resulted in detection of as few as 500 labelled cells in vivo. Two weeks after stereotactic transplantation, the luciferase signal was sustained traceable, with approximately 18% of the original luciferase signal detectable for monocytes and about 30% for macrophages. Hypointensity in MRI of the graft appeared unaltered in spatial location. In a therapeutically relevant approach, systemic cell administration after stroke resulted in accumulation mostly in thoracic regions, as could be visualized with BLI. For detection of homing to ischemic brain tissue more cells need to be administered. Nevertheless, during parallel MRI sessions recruitment of i.v. injected cells to the lesion site could be detected by day 2 post stroke as scattered hypointense signal voids. With further increase in sensitivity, our multi-facetted labelling strategy will provide the basis for in vivo tracking and fate specification of tissue-infiltrating macrophages and their distinct role in stroke-related neuro-inflammation.

No MeSH data available.


Related in: MedlinePlus