Experimental model of warfarin-associated intracerebral hemorrhage

Abstract

Background and purpose: Future demographic changes predict an increase in the number of patients with atrial fibrillation. As long-term anticoagulation for the prevention of ischemic strokes becomes more prevalent, the burden of warfarin-associated intracerebral hemorrhage (W-ICH) is likely to grow. However, little is known about the clinical aspects and pathophysiologic mechanisms of W-ICH. This study describes the development of a mouse model of W-ICH in which hematoma growth and outcomes can be correlated with anticoagulation parameters.

Methods: CD-1 mice were treated with warfarin (2 mg/kg per 24 hours) added to drinking water. ICH was induced by stereotactic injection of collagenase type VII (0.075 U) into the right striatum. Hemorrhagic blood volume was quantified by means of a photometric hemoglobin assay 2 and 24 hours after hemorrhage induction. Neurologic outcomes were assessed on a 5-point scale.

Results: The international normalized ratio in nonanticoagulated mice was 0.8+/-0.1. After 24 (W-24) and 30 (W-30) hours of warfarin pretreatment, international normalized ratio values increased to 3.5+/-0.9 and 7.2+/-3.4, respectively. Compared with nonanticoagulated mice, mean hemorrhagic blood volume determined 24 hours after hemorrhage induction was found to be 2.5-fold larger in W-24 mice (P=0.019) and 3.1-fold larger in W-30 mice (P<0.001, n=10 per group). Mortality at 24 hours after hemorrhage induction was 0% in nonanticoagulated mice, 10% in W-24 mice, and 30% in W-30 mice. Hematoma enlargement between 2 and 24 hours after hemorrhage induction was -1.4% for nonanticoagulated mice, 22.9% for W-24 mice, and 62.2% for W-30 mice.

Conclusions: This study characterizes the first experimental model of W-ICH. It may be helpful in gaining further insights into the pathophysiology of W-ICH and may be used for testing the efficacy of treatment strategies, such as hemostatic therapy, in this severe subtype of stroke.

Figure 1

INR values before and after 24 hours (left) and 30 hours (right) of warfarin administration in drinking water. INR values were also determined 6 and 24 hours after withdrawal of warfarin administration.

Figure 2

Experimental study protocol. C indicates control mice (no warfarin pretreatment); W-24, mice with 24 hours of warfarin feeding in drinking water; and W-30, mice with 30 hours of warfarin feeding in drinking water. ICH induction is the time point of ICH induction via stereotactic administration of collagenase into the right striatum. In all 3 groups, neurologic outcome, hemorrhagic blood volume (HBV), and hematoma size (HS) were determined 2 and 24 hours after hemorrhage induction.

Figure 3

Left, Representative brain slices of the needle track 24 hours after sham operation (ie, administration of 0.5 µL saline into the right striatum) in C mice as well as in mice after 24 (W-24) and 30 (W-30) hours of warfarin pretreatment. Right, Representative brain slices 24 hours after hemorrhage induction by collagenase administration into the right striatum. In anticoagulated mice, the hematoma appeared to be slightly larger but much more dense compared with that in C mice. W-30 mice had amounts of still-noncoagulated blood.

Figure 4

Left, Hemorrhagic blood volume measured 2 and 24 hours after hemorrhage induction by quantitative hemoglobin content determination in C mice and in mice with 24 (W-24) and 30 (W-30) hours of warfarin pretreatment. Right, Hematoma size measured 2 and 24 hours after hemorrhage induction by computer-assisted outlining of brain slices in C, W-24, and W-30 mice.

Figure 5

A, Neurologic outcome in C mice and in mice with 24 (W-24) and 30 (W-30) hours of warfarin pretreatment, as assessed on a 5-point scale. B, Results of the hanging wire test. The time period to falloff was recorded, and a maximum of 60 seconds of hanging was allowed. C, Percentage of body weight loss 24 hours after hemorrhage induction or sham operation.