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ICAM-1null C57BL/6 Mice Are Not Protected from Experimental Ischemic Stroke

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Abstract

Accumulation of neutrophils in the brain is a hallmark of cerebral ischemia and considered central in exacerbating tissue injury. Intercellular adhesion molecule (ICAM)-1 is upregulated on brain endothelial cells after ischemic stroke and considered pivotal in neutrophil recruitment as ICAM-1-deficient mouse lines were found protected from experimental stroke. Translation of therapeutic inhibition of ICAM-1 into the clinic however failed. This prompted us to investigate stroke pathogenesis in Icam1tm1Alb C57BL/6 mutants, a true ICAM-1null mouse line. Performing transient middle cerebral artery occlusion, we found that absence of ICAM-1 did not ameliorate stroke pathology at acute time points after reperfusion. Near-infrared imaging showed comparable accumulation of neutrophils in the ischemic hemispheres of ICAM-1null and wild type C57BL/6 mice. We also isolated equal numbers of neutrophils from the ischemic brains of ICAM-1null and wild type C57BL/6 mice. Immunostaining of the brains showed neutrophils to equally accumulate in the leptomeninges and brain parenchymal vessels of ICAM-1null and wild type C57BL/6 mice. In addition, the lesion size was comparable in ICAM-1null and wild type mice. Our study demonstrates that absence of ICAM-1 neither inhibits cerebral ischemia-induced accumulation of neutrophils in the brain nor provides protection from ischemic stroke.

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Acknowledgements

We would like to thank the Microscopy Imaging Center (MIC) of the University of Bern, especially Dr. Roch-Philippe Charles (IBMM), for support in image analysis. We express our sincere thanks to Claudia Blatti, Therese Périnat, and Albert Witt for technical help in this study. We thank Dr. Urban Deutsch for help with the mouse breeding and genotyping logistics. Additional thanks go to Katrin Bissegger, Isabelle Wymann, and Svetlozar Tsonev for professional caretaking of the mice.

Author Contribution Statement

G.E. and M.V. performed experiments including surgeries and data analysis, and G.E. wrote the manuscript. S.P. performed the experiments, analyzed the data, and prepared some figures. B.E. and J.K. outlined and supervised the project, evaluated data, and edited the manuscript.

Funding

This study was funded by the EU FP7 European Stroke Network (grant nos. 201024 and 202213 to BE), the Swiss Heart Foundation (to BE and GE), and the Swiss National Science Foundation (grant PZ00P3_136822), and Hartmann-Müller Foundation (to JK).

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Authors and Affiliations

  1. Theodor Kocher Institute, University of Bern, Freiestrasse 1, 3012, Bern, Switzerland

    Gaby U. Enzmann, Sofia Pavlidou & Britta Engelhardt

  2. Institute for Biomedical Engineering, ETH and University of Zurich, 8093, Zurich, Switzerland

    Markus Vaas & Jan Klohs

  3. Neuroscience Center Zurich, University of Zurich and ETH Zurich, Zurich, Switzerland

    Markus Vaas & Jan Klohs

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  1. Gaby U. Enzmann
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  2. Sofia Pavlidou
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Correspondence to Britta Engelhardt.

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Ethical Approval

All applicable international, national, and institutional guidelines for the care and use of animals were followed. In detail, in vivo experiments were approved by the Veterinary Office of the Canton of Bern (Switzerland) (licenses BE79/11, BE127/14). Animal research was performed in accordance to the Swiss legislation on the protection of animals and in compliance with the ARRIVE guidelines for reporting animal research (https://www.nc3rs.org.uk/arrive-guidelines).

Conflict of Interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Suppl. Fig. 1

Absence of ICAM-1 does not induce compensatory upregulation of endothelial cell adhesion molecules in the ischemic hemisphere following 60-min ischemia and 24-h reperfusion. ICAM-1null and wild type (WT) C57BL/6 mice displayed numerous VCAM-1 positive blood vessels both in the ipsilateral cortex (Fig. 1(a’, d’)) and in the meningeal compartment (Fig. 1(c’, f’)). In relation to ICAM-1-positive blood vessels in WT C57BL/6 mice (Fig. 1(a–c)), VCAM-1-positive vessels outnumbered the former and were distributed rather evenly throughout the lesioned hemisphere. Lack of ICAM-1 immunoreactivity in ICAM-1null C57BL/6 mice confirmed the complete absence of ICAM-1 isoforms (Fig. 1(d–f)). We observed de novo synthesis of P-selectin in parenchymal microvessels (Fig. 1(a”–b”, e”–f”)) in C57BL/6 mice of either genotype. The scale bar equals 50 μm and applies to all images (GIF 689 kb)

High-resolution image (TIFF 3465 kb)

Suppl. Fig. 2

ICAM-1 is dispensable for neutrophil accumulation in blood vessels following stroke and reperfusion. Double immunofluorescence staining of neutrophils (Ly6G, green) and ICAM-1 (red) revealed that ICAM-1 is not required for localization of neutrophils to microvessels in the ischemic hemisphere. Both ICAM-1-positive brain microvessels (wild type (WT) mouse, left column, arrow) and blood vessels deficient of ICAM-1 (ICAM-1null, right column, arrowheads) permitted neutrophil adhesion. The scale bar equals 50 μm (GIF 40 kb)

High-resolution image (TIFF 9147 kb)

Suppl. Fig. 3

VCAM-1 does not exclusively mediate neutrophil adhesion to brain microvessels after stroke. Double immunofluorescence staining of neutrophils (Ly6G, green) and VCAM-1 (red) confirmed neutrophil endothelial interaction in both the presence (arrow) and absence (arrowhead) of VCAM-1 following transient ischemia and reperfusion in wild type (WT) (a–d) and ICAM-1null (a’–d’) C57BL/6 mice. The scale bar equals 50 μm (GIF 54 kb)

High-resolution image (TIFF 6053 kb)

Suppl. Fig. 4

Expression of P-selectin does not favor neutrophil localization to brain microvessels after stroke. Double immunofluorescence staining of neutrophils (Ly6G, green) and P-selectin (red) demonstrated no preferential localization of neutrophils to P-selectin positive microvessels in the ischemic cortex of wild type (WT) (left column) and ICAM-1null (right column) C57BL/6 mice. Neutrophils localized to the perivascular compartment in both the presence (white arrow) and absence (white arrowhead) of P-selectin. Occasionally, neutrophils were observed adjacent to platelets, which express P-selectin as well (yellow arrow). Irrespective of the expression of P-selectin, neutrophils were also detected in the leptomeningeal compartment (yellow arrowhead). The scale bar equals 50 μm (GIF 84 kb)

High-resolution image (TIFF 5633 kb)

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Enzmann, G.U., Pavlidou, S., Vaas, M. et al. ICAM-1null C57BL/6 Mice Are Not Protected from Experimental Ischemic Stroke. Transl. Stroke Res. 9, 608–621 (2018). https://doi.org/10.1007/s12975-018-0612-4

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