Blocking of platelet glycoprotein receptor Ib reduces "thrombo-inflammation" in mice with acute ischemic stroke

Michael K Schuhmann, Josua Guthmann, Guido Stoll, Bernhard Nieswandt, Peter Kraft, Christoph Kleinschnitz, Michael K Schuhmann, Josua Guthmann, Guido Stoll, Bernhard Nieswandt, Peter Kraft, Christoph Kleinschnitz

Abstract

Background: Ischemic stroke causes a strong inflammatory response that includes T cells, monocytes/macrophages, and neutrophils. Interaction of these immune cells with platelets and endothelial cells facilitates microvascular dysfunction and leads to secondary infarct growth. We recently showed that blocking of platelet glycoprotein (GP) receptor Ib improves stroke outcome without increasing the risk of intracerebral hemorrhage. Until now, it has been unclear whether GPIb only mediates thrombus formation or also contributes to the pathophysiology of local inflammation.

Methods: Focal cerebral ischemia was induced in C57BL/6 mice by a 60-min transient middle cerebral artery occlusion (tMCAO). Animals were treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab). Rat immunoglobulin G (IgG) Fab was used as control treatment. Stroke outcome, including infarct size and functional deficits as well as the local inflammatory response, was assessed on day 1 after tMCAO.

Results: Blocking of GPIb reduced stroke size and improved functional outcome on day 1 after tMCAO without increasing the risk of intracerebral hemorrhage. As expected, disruption of GPIb-mediated pathways in platelets significantly reduced thrombus burden in the cerebral microvasculature. In addition, inhibition of GPIb limited the local inflammatory response in the ischemic brain as indicated by lower numbers of infiltrating T cells and macrophages and lower expression levels of inflammatory cytokines compared with rat IgG Fab-treated controls.

Conclusion: In acute ischemic stroke, thrombus formation and inflammation are closely intertwined ("thrombo-inflammation"). Blocking of platelet GPIb can ameliorate thrombo-inflammation.

Keywords: Glycoprotein receptor Ib; Ischemic stroke; Thrombo-inflammation; Transient middle cerebral artery occlusion.

Figures

Fig. 1
Fig. 1
Infarct size, neurologic scoring, and brain microvascular thrombosis 24 h after stroke induction. a Representative TTC stainings of three corresponding coronal brain sections of mice treated with rat IgG Fab (Ctrl Fab) or p0p/B Fab (GPIb) 24 h after induction of tMCAO. Ischemic infarctions appear white, while vital tissue is stained red. b Infarct volumes as measured by planimetry (n = 8–10/group). c Bederson score and d Grip test score 24 h after tMCAO (n = 8–10/group). e Representative immunocytologic stainings of platelet aggregates within the vasculature from the ipsilateral hemisphere of mice, treated with rat IgG Fab (Ctrl Fab) or p0p/B Fab (GpIb) on day 1 after tMCAO. Hoechst depicts cell nuclei, CD31 stains endothelial cells, and GPIX represents platelet aggregates. Scale bar, 10 μm. f Quantification of ipsilesional glycoprotein IX (GPIX)-positive aggregates in GPIb-treated mice when compared with control mice. (n = 5 or 6/group). g Quantification of occluded ipsilesional vessels in hematoxylin–eosin (H&E)-stained brain sections on day 1 after tMCAO (n = 5–6/group). b, f, g, *P < 0.05, **P < 0.01, unpaired Student’s t test. c, d, *P < 0.05, Mann–Whitney U test
Fig. 2
Fig. 2
Quantification of immune cell accumulation and relative cytokine gene expression in the infarcted hemisphere at day 1 after stroke. a Representative CD11b-immunoreactivity 24 h after tMCAO of mice treated with rat IgG Fab (Ctrl Fab) or p0p/B Fab (GPIb). Scale bar, 100 μm. c Schematic view of the brain regions analyzed to quantify the density of immune cells per mm2. b, d, e Quantification of CD11b-positive cells per slice and CD11b-positive cells/mm2 in different cortical and basal ganglial regions in the ipsilateral hemisphere at day 1 (n = 5/group). f Representative immunocytologic stainings of brain-infiltrating CD3-positive T lymphocytes on day 1 after tMCAO of mice, treated with rat IgG Fab (Ctrl Fab) or p0p/B Fab (GPIb). Scale bar, 25 μm. g–i Quantification of CD3-positive cells per slice and CD3-positive cells/mm2 in different cortical and basal ganglial regions in the ipsilateral hemisphere at day 1 (n = 5/group). j, k Relative gene expression of tumor necrosis factor-α (TNFα), interleukin-1β (IL1β), and interleukin-6 (IL6) in the cortical and basal ganglia ischemic hemispheres of mice, treated with rat IgG Fab (Ctrl Fab) or p0p/B Fab (GPIb) (n = 6/group). *P < 0.05, ***P < 0.001, unpaired Student’s t test

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Source: PubMed

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