Elsevier

NeuroImage

Volume 49, Issue 4, 15 February 2010, Pages 2907-2914
NeuroImage

Enhanced cortical reperfusion protects coagulation factor XII-deficient mice from ischemic stroke as revealed by high-field MRI

https://doi.org/10.1016/j.neuroimage.2009.11.061Get rights and content

Abstract

Intrinsic coagulation factor XII deficient (FXII−/−) mice are protected from ischemic stroke. To elucidate underlying mechanisms we investigated the early ischemic period in vivo by multimodal magnetic resonance imaging (MRI) at 17.6 Tesla.

Cerebral ischemia was induced by either transient (60 min) or permanent occlusion of the middle cerebral artery (t/pMCAO). 10 FXII/ mice underwent t- , 10 FXII/ mice p- and 10 Wildtype (Wt) mice tMCAO. Cerebral blood flow (CBF), diffusion-weighted-imaging (DWI) and T2-relaxometry were measured at 2 h and 24 h after MCAO. Outcome measures were evaluated after motion correction and normalization to atlas space. 2 h after tMCAO CBF reduction was similar in FXII/ and Wt mice extending over cortical (CBF (ml/100 g/min) 33.6 ± 6.9 vs. 35.3 ± 4.6, p = 0.42) and subcortical regions (25.7 ± 4.5 vs. 31.6 ± 4.0, p = 0.17). At 24 h, recovery of cortical CBF by +36% was observed only in tMCAO FXII/ mice contrasting a further decrease of – 30% in Wt mice after tMCAO (p = 0.02, F(1,18) = 6.24). In FXII/ mice in which patency of the MCA was not restored (pMCAO) a further decrease of − 75% was observed. Cortical reperfusion in tMCAO FXII/ mice was related to a lower risk of infarction of 59% vs. 93% in Wt mice (p = 0.04). Subcortical CBF was similarly decreased in both tMCAO groups (Wt and FXII/) relating to a similar risk of infarction of 89% (Wt) vs. 99% (FXII/, p = 0.17).

Deficiency of FXII allows neocortical reperfusion after tMCAO and rescues brain tissue by this mechanism. This study supports the concept of FXII as a promising new target for stroke prevention and therapy.

Introduction

Thromboembolic occlusion of cerebral vessels accounts for about two thirds of ischemic strokes representing one of the leading causes of mortality and chronic disability worldwide (Murray and Lopez, 1997). In acute human thromboembolic stroke rapid recanalization of major arteries represents the principal goal of treatment (Davis et al., 2008). However, it is an unfortunate clinical observation that despite reopening of a previously occluded major cerebral artery, referred to as “recanalization,” further infarct growth may occur (Coutts and Goyal, 2009, Soares et al., 2009, Yoo et al., 2009). This may be due to distal thrombus migration or reocclusion, but also occurs in vascular territories of persistently reopened major cerebral arteries. In experimental observations in the stroke model of transient middle cerebral artery occlusion (tMCAO), microvascular obstruction, also termed focal “no-reflow,” early after the ischemic stimulus is related to the failure of efficient brain tissue reperfusion despite recanalization of major intracerebral branches. Among other important mechanisms, the plasmatic coagulation system (Okada et al., 1994) and activation of platelets (Choudhri et al., 1998) mediate microvascular obstruction and may be potentially important secondary steps in stroke development (reviewed in (Stoll et al., 2008)), which, however, remains to be proven in human stroke.

We have recently shown that coagulation factor XII (FXII; Hageman factor) is involved in thrombus formation after transient middle cerebral artery occlusion (tMCAO), the most widely used stroke model (Kleinschnitz et al., 2006). For initiating plasmatic coagulation two distinct pathways exist, either triggered by vessel wall (extrinsic) or by blood-borne (intrinsic) factors, and converge on a common pathway leading to thrombin and fibrin formation. The intrinsic pathway of coagulation is initiated when FXII comes into contact with negatively charged surfaces (contact activation) and plays a central role in pathological thrombus formation in various vessel injury models. Importantly, FXII is dispensable for hemostasis after tissue injury (Renne et al., 2005). In the tMCAO stroke model FXII-deficient (FXII/) mice developed smaller infarcts as revealed by histology (Kleinschnitz et al., 2006) but the precise mechanisms of this stroke protection are unknown. To further address this important functional issue we employed a multimodal MRI protocol at high-field strength of 17.6 Tesla, and followed the early phase of cerebral ischemia and the evolution of infarctions in vivo in individual animals over time. FXII/ mice underwent transient (t) or permanent (p) MCAO. MRI outcome measures were chosen to characterize cerebral perfusion, hypoxic diffusion restriction by diffusion weighted imaging (DWI), and irreversible infarction as assessed by lesion extent on T2-weighted MRI. Cerebral perfusion as the principal outcome measure was determined as cerebral blood flow (CBF) in the brain microvasculature using water as a freely diffusible tracer by a modified continuous arterial spin labeling (CASL) method (Detre et al., 1992, Williams et al., 1992, Wong et al., 1998). For an exact spatial allocation of all outcome measures to relevant anatomical structures in the mouse brain image registration was performed to a standard mouse brain atlas space. To match responses over time and between groups with sufficient anatomical detail a large sample size was obtained.

With this design we for the first time followed stroke development in vivo in a transgenic mouse model using FXII/ mice by multimodal high field MRI. Our data indicate that CBF is similarly reduced in FXII/ and Wildtype (Wt) mice early after tMCAO, but recovers in the cortex of FXII/ animals at later time points leading to smaller infarctions. These findings provide important insights into thrombus formation after experimental stroke and indicate that enhanced tissue reperfusion is one major mechanism that protects FXII/ mice from ischemic stroke. Importantly, because targeting FXII seems not to be related to an increased risk of bleeding complications, it seems promising in human ischemic stroke for the prevention of postischemic tissue damage.

Section snippets

Experimental design and animal stroke model

All procedures and animal studies were approved by the Regierung von Unterfranken (Würzburg, Germany) and conducted in accordance with the recommendations for the performance of basic experimental stroke studies as previously published (Dirnagl, 2006). The generation of FXII/ mice has been described in detail previously (Pauer et al., 2004).

The experimental group in this study were FXII−/− mice undergoing 60 min. occlusion of the MCA (tMCAO FXII/; N = 10). To control for the effect of FXII/

Hemodynamic response in Wildtype and FXII/ mice after transient and permanent MCAO

In Wt mice initial hypoperfusion further deteriorated in the cortex and subcortex from 2 h to 24 h after tMCAO, reflected by the significant cluster of perfusion deactivation overlayed in blue (Fig. 1, contrast 2 h  >  24 h for Wt after tMCAO).

In contrast, in FXII/ mice the initial hypoperfusion recovered in the cortex from 2 h to 24 h after tMCAO, as indicated by the significant cluster of perfusion activation overlayed in yellow (Fig. 1, contrast 2 h < 24 h for FXII/ mice after tMCAO).

Discussion

The cellular and molecular mechanisms that contribute to ongoing thrombus formation and tissue damage, despite recanalization of a previously occluded major cerebral artery and reperfusion after cerebral ischemia, are incompletely understood, but of outmost clinical importance (Stoll et al., 2008). We have recently demonstrated by 1.5 T MRI and histopathological analysis that deficiency or pharmacological blockade of the intrinsic pathway of coagulation, e.g. FXII or FXI, protects mice from

Acknowledgments

This study was supported by the Deutsche Forschungsgemeinschaft, Bonn Sonderforschungsbereich 688 A3,B1) and an endowed professorship for neuroimaging to M.B. donated by Schering AG, Berlin to the University of Würzburg. We thank Peter Kraft (University of Würzburg, Würzburg, Germany) for technical assistance in conducting the experimental procedures.

References (28)

  • DirnaglU.

    Bench to bedside: the quest for quality in experimental stroke research

    J. Cereb. Blood Flow Metab.

    (2006)
  • HerscovitchP. et al.

    What is the correct value for the brain–blood partition coefficient for water?

    J. Cereb. Blood Flow Metab.

    (1985)
  • KleinschnitzC. et al.

    Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis

    J. Exp. Med.

    (2006)
  • KleinschnitzC. et al.

    Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding

    Circulation

    (2007)
  • Cited by (0)

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    Contributed equally to this study.

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