Abstract
Previous reports have suggested that direct oral anticoagulants exert a prothrombolytic
effect against intracardiac thrombi. We hypothesized that these anticoagulants may also
help recanalize occluded intracranial arteries via prothrombolytic effects. In this study,
we evaluated the effects of rivaroxaban, a direct oral anticoagulant, on fibrin emboli
within the cerebrocortical microvessels in a mouse model of embolic stroke. Fibrin emboli
prepared ex vivo were injected into the common carotid artery of male
C57BL/6 mice, and embolization in the microvessels on the brain surface was observed
through a cranial window. Oral administration of rivaroxaban was initiated a week before
injection of the emboli. The number and sizes of the emboli were measured at two time
points: immediately after and 3 h after the embolus injection in the rivaroxaban-treated
mice (n =6) and untreated mice (n =7). The rates of recanalization and change in the
embolus size were analyzed between the two groups. Complete recanalization was observed
only in the rivaroxaban group (three mice in the rivaroxaban group compared with none in
the control group). A significantly higher rate of reduction of the embolus size was
observed in the rivaroxaban group than in the control group (P=0.0216).
No significant differences between the two groups were observed in the serum levels of the
following coagulation markers: thrombin–antithrombin III complexes, D-dimers, or
plasmin–α2-plasmin inhibitor complex. Our findings indicate that rivaroxaban may promote
reduction in the size of stagnated fibrin emboli in cerebrocortical microvessels in cases
of embolic stroke.
Introduction
Direct oral anticoagulants (DOACs) have recently proven effective for preventing
cardioembolic stroke in patients with nonvalvular atrial fibrillation (NVAF).1 Moreover, DOACs are widely preferred over
warfarin potassium. Typically, anticoagulation therapies, including DOACs and warfarin
potassium, are used to prevent recurrent cardioembolic stroke.1,2
However, some reports have suggested that DOACs may also exert a prothrombolytic effect
against intracardiac thrombi.3,4 Inhibition
of thrombin activatable fibrinolysis inhibitor (TAFI), which can be activated by thrombin
and thrombomodulin, is likely one mechanism underlying the prothrombolytic effect of
DOACs.5 From these findings, we
speculated that DOACs, via prothrombolytic effects, may help recanalize intracranial
arteries that have been occluded by cardiogenic fibrin emboli.
Several studies have suggested that improved clinical recovery occurs in patients with
subacute recanalization of the occluded arteries by rescue of the penumbra, an unstable
ischemic region around the ischemic focus.6,7,8,9
Nevertheless, initiation of anticoagulant treatment in the acute phase of ischemic stroke is
generally not recommended because of the risk of intracranial bleeding events.10,11 However, DOACs may be administered safely even in the acute
phase of ischemic stroke because of the lower risk of bleeding events compared to the risks
associated with warfarin potassium.1,12,13
Considering the greater safety and possible local prothrombolytic effects of DOACs, we
hypothesized that, in cases of acute embolic stroke, early initiation of DOAC administration
may promote reduction of embolus size, with only a low risk of intracranial hemorrhagic
events. In the current study, we evaluated the effects of rivaroxaban, a DOAC that inhibits
factor Xa, on fibrin emboli within the cerebrocortical microvessels in a mouse model of
embolic stroke.
Materials and Methods
Animals
The experimental protocol was approved by the Ethics Committee on Animal Care and Use,
Keio University (#09058). The male C57BL/6 mice aged 9–11 weeks used in this study were
purchased from CLEA Japan Inc. (Tokyo, Japan).
Anesthesia
All animal procedures were performed with the animals under general anesthesia induced by
2.0% isoflurane (Pfizer, New York, NY, USA) and maintained with 1.5%–1.7% isoflurane mixed
with room air. No artificial mechanical ventilation was used during the experiments.
Cranial window
A cranial window was created using a previously described method.14 Briefly, using an operating microscope (S21;
Carl Zeiss, Oberkochen, Germany), the cranial bone was drilled and thinned above the left
parieto-occipital cortex, 4.0 mm posterior to the bregma and 4.0 mm to the left of the
midline, until the brain parenchyma and vessels could be observed through the thinned bone
and dura. The diameter of the cranial window was approximately 3.0 mm. After creating the
window, we dripped oil on this segment and sealed it with a circular 140-μm-thick quartz
coverslip using cement (Fig. 1A). A metal bar
was attached to the skull of the mouse to enable multiple observations of the same site in
the same animal. Microvessels on the brain surface could be observed through the cranial
window under a fluorescence microscope equipped with an Hg lamp (LH-M100CB-a; Nikon Co.,
Tokyo, Japan) and a band-pass filter (G2A, Nikon Co.) (Fig. 1B).
Fibrin emboli
Fibrin emboli were prepared ex vivo using a previously reported method
with modification.15 Briefly, 10 mg of
human fibrinogen (Sigma-Aldrich Co.; St Louis, MO, USA), 0.2 units of human thrombin
(Sigma-Aldrich Co.), and 6 mg of rhodamine B isothiocyanate-dextran 70 (RITC-dextran;
8 mg/ml; Sigma-Aldrich Co.) were mixed with 1 mL of normal saline, and the mixture was
refrigerated at 4°C overnight. Before injection of the embolic mixture on the following
day, the fibrin emboli were homogenized using 0.5 mL normal saline and then passed through
a hydrophilic nylon net filter (pore size, 41.0 μm; Merck Millipore Ltd., Burlington, MA,
USA). The fibrin emboli could be visualized under a fluorescence microscope (Fig. 2).
Rivaroxaban administration and analysis of blood concentrations
We divided the mice into two groups: the rivaroxaban group and the control group. The
mice were housed under a 12-h day/night cycle. Administration of laboratory chow, either
mixed or not mixed with rivaroxaban (1200 ppm), was initiated 7 days before injection of
the embolic mixture. To confirm the blood concentrations of rivaroxaban, 12 mice (control
group, n=6; rivaroxaban group, n=6), in addition to the mice that were used for the
embolic mixture injection experiment, were sacrificed 7 days after the initiation of
feeding (according to a schedule similar to that in the embolic mixture injection
experiment), and blood samples were collected from the heart. The blood samples were
centrifuged at 2000 rpm for 5 min to obtain serum samples. The rivaroxaban concentrations
in these serum samples were determined using liquid chromatography (Shin Nippon Biomedical
Laboratories Ltd., Tokyo, Japan).
Injection of the embolic mixture
The experimental protocol is shown in Fig. 3.
We used 16 mice (9 mice in the control group and 7 mice in the rivaroxaban group) for this
experiment and compared data between 7 mice in the control group and 6 mice in the
rivaroxaban group, as described in the “Results” section. The cranial window was created
at approximately 10:00 pm on the night before injection of the embolic mixture.
Simultaneously, we also prepared the embolic mixture by mixing fibrinogen, thrombin, and
rhodamine dextran and kept the mixture under refrigeration. At 8:00–9:00 am on the
following morning, the mice were anesthetized and placed in the supine position. A
catheter was then inserted into the left common carotid artery (CCA). The animals were
subsequently placed in the prone position so that the brain surface could be
microscopically observed through the cranial window. We then injected 0.1 mL of
homogenized embolic mixture through the catheter and confirmed the presence of stagnated
emboli in the arteries on the brain surface by observation with a fluorescence microscope
through the cranial window. If stagnated emboli were not identified after the first
injection, further 0.05-mL injections (up to a maximum of four additional injections) were
administered until stagnated emboli were seen. After confirmation of emboli, we removed
the catheter, closed the skin incision, and left the mice in their home cages with free
access to water and food. Three hours later, the mice were anesthetized again and held in
place by the metal bar so that the same site of the brain surface could be observed once
again through the cranial window.
Images observed through the cranial window were captured by a video camera attached to
the fluorescence microscope at two time points: immediately after and 3 h after injection
of the embolic mixture. Emboli labeled with rhodamine were detected under an Hg lamp, and
the other nonfluorescent structures were evident in the bright-field images.
The total number of emboli observed through the cranial window immediately after
injection of the emboli mixture was counted by three examiners who were blinded to the
groups to which the animals had been allocated (rivaroxaban/control). The number of emboli
that were still observable in the same locations 3 h after injection of the emboli mixture
was also counted by the three blinded examiners. The rate of recanalization was calculated
according to the following formula:
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Additionally, we measured the sizes of the emboli at each time point in each animal using
Image J,16 which is free calculation
software. The sum of the sizes of all the emboli visualized through the cranial window was
measured by three blinded examiners. The sum of the sizes of the emboli th were still
observable at the same locations 3 h after injection of emboli was again measured by the
three examiners. The rate of change of the embolus size was calculated according to the
following formula:
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The median value of the data counted/measured by the three examiners was adopted for all
image analyses.
Measurements of coagulation markers in blood
We obtained serum samples from 10 mice that were not part of the injected thrombi
experiments (control group, n=5; rivaroxaban group, n=5) to measure the blood levels of
coagulation and thrombolysis markers, i.e., thrombin–antithrombin III complex (TAT),
D-dimers, and plasmin–α2-plasmin inhibitor complex (PIC). To reproduce the conditions, all
procedures performed on the mice in the embolic mixture injection experiment were also
performed on this group. The mice were sacrificed 3 h after injection of the embolic
mixture. Analysis of the blood samples was outsourced to SRL Inc., Tokyo, Japan.
Statistical analysis
The Mann–Whitney U test was used to compare data between the groups. P
< 0.05 was considered statistically significant. All statistical analyses were
performed using GraphPad PRISM version 6 (GraphPad Software, Inc. CA, USA).
Results
Blood concentrations of rivaroxaban
After administration of rivaroxaban for 7 days, the blood concentrations of rivaroxaban
in 6 mice (the rivaroxaban group) were in the range of 0.292–1.25 µg/ml (mean,
0.662 µg/ml), which closely matched the adequate range, 0.4–1.0 µg/ml in daylight, as
previously determined by Bayer Pharma AG, a manufacturer of rivaroxaban. (This adequate
range was obtained using animals administered with a chow diet containing 1200 ppm
rivaroxaban; this drug concentration was determined to inhibit clot formation by >70%
in the rodent venous thromboembolism model).17 Conversely, the blood level of rivaroxaban was below the detection
limit in the control group (n=6) fed with normal laboratory chow for 7 days.
Observation of injected emboli through the cranial window
In the current experiment, there was possibly a difference in the duration of laser
exposure between the two groups, and this difference could potentially influence the
result through reduced intensity of the fluorescence of emboli. However, in an ex
vivo study, we confirmed that the size of emboli did not change under
continuous laser exposure for 5 min (data not shown); the total observation time under a
fluorescence microscope for the two time points in the current experiment was typically
less than 2 min.
Before they were injected, the diameters of the emboli were measured under a fluorescence
microscope, and the mean ± SD was 13.2 ± 5.0 µm. A recently published article from our
group stated that artificial microspheres with a diameter ranging from 13 to 24 µm could
distribute through the arteries in the cortex.18 Consequently, the size of the emboli prepared using our method was
considered suitable for observing emboli in the microvessels on the brain surface.
After injection via the CCA, the embolic mixture labeled with rhodamine traveled through
the arteries and finally coalesced and stagnated in the microvessels on the brain surface.
The resulting stagnated emboli were successfully visualized through the cranial window
using a camera attached to the fluorescence microscope (Fig. 4A–H, a supplementary video is also available).
Calculation of the recanalization rates and rates of change in embolus size
Another batch of mice was included in this study (nine mice in the control group and
seven mice in the rivaroxaban group). One mouse in the control group and one mouse in the
rivaroxaban group died during the interval between injection of the embolic mixture and
the observation performed 3 h after the injection. Another mouse in the control group was
excluded because no stagnated emboli could be observed through the cranial window, even
after four additional embolus injections. Therefore, seven mice in the control group and
six mice in the rivaroxaban group finally underwent calculation of the recanalization rate
and the rate of change in emboli size. [In most cases, the stagnated emboli could be
visualized after a single injection (0.1 mL) of the embolic mixture. However, three mice
in the rivaroxaban group and three mice in the control group required a single additional
injection (0.05 mL of the mixture) for successful visualization of emboli].
At 3 h after emboli were injected, new emboli were occasionally observed at sites where
emboli were not observed immediately after the injection of emboli. We speculated that
these emboli were located at the proximal parts of arteries that were initially outside
the field of view through cranial windows. These emboli then traveled to the area where
they could be observed through the cranial window 3 h after injection. However, it was
difficult to confirm this hypothesis. Moreover, it was impossible to calculate the
chronological change in each embolus when we included these newly observed emboli in the
analysis. Therefore, we focused only on the emboli that were still visible in the same
location at 3 h after injection of emboli.
The median (interquartile range) of the recanalization rate in the control group and
rivaroxaban group were 43.3% (16.7%–66.7%) and 75.0% (20.0%–100.0%), respectively
(Fig. 4I). This difference was not significant between the groups
(P=0.3077). However, mice with complete (100%) recanalization were
observed only in the rivaroxaban group (three of six mice), and no mice with complete
recanalization were observed in the control group (none of seven mice).
In the detailed observation of emboli, some did not disappear but had decreased in size
by 3 h after the injection (representative images are shown in Fig. 4C,D). This change was recognized as “partial recanalization
at each occluded site” and is important in clinical situations. However, the analysis of
the recanalization rate did not reflect these partial changes. Therefore, we proceeded to
analyze the embolus size. First, the embolus size immediately after injection did not
differ between the two groups (P=0.3566; Fig. 4J). This
finding confirmed that the fibrin emboli prepared ex vivo were not
influenced by administration of rivaroxaban prior to the emboli injection. At 3 h after
injection of the embolic mixture, the total embolus size was significantly smaller in the
rivaroxaban group than in the control group (P=0.0344, Fig.
4K). Moreover, the rivaroxaban group had a significantly higher rate of decrease
of the embolus size than the control group did (P=0.0216). The median
(interquartile range) rates of change in the control group and rivaroxaban group were
52.5% (46.3%–81.9%) and 18.7% (0.0%–48.5%), respectively. These data suggested that
rivaroxaban promoted the size reduction of emboli within cerebrocortical microvessels.
Analysis of coagulation markers in blood
To investigate the mechanism underlying the decrease in embolus size in the rivaroxaban
group, the blood levels of coagulation markers TAT, PIC, and D-dimers were measured. The
levels of TAT in the control group and rivaroxaban group were not significantly different
(2.8 ± 1.7 vs. 2.3 ± 1.7 ng/ml; mean ±SD), whereas the levels of PIC and D-dimers were
below the respective detection limits in both groups.
Discussion
We successfully observed stagnated fibrin emboli in the cerebral microvessels on the brain
surface through a cranial window created in a mouse model of embolic stroke after injection
of an embolic mixture into the CCA. Furthermore, the rate of reduction of the embolus size
was significantly higher in the rivaroxaban group than in the control group.
Several studies have described a mouse model of embolic stroke using fibrin-rich clots
labeled with fluorescents.15,19,20 We
combined this embolic stroke model with the creation of a cranial window that allowed direct
visualization of the stagnated fibrin emboli in the cerebral microvessels on the brain
surface. As a result, our model could provide in vivo imaging and multiple
chronological observations of the same area on the brain surface in the same animal, which
is useful, for example, to investigate the effects of drugs in vivo.
We propose the following two mechanisms to explain the decrease in embolus size in the
rivaroxaban group. The first is that rivaroxaban inhibits secondary thrombus formation by
exerting an anticoagulant effect. This effect results in the relative dominance of the
endogenic thrombolytic mechanism, which ultimately leads to earlier lysis of thrombus. The
second is that rivaroxaban itself exerts a prothrombolytic effect.
Because rivaroxaban exerts its anticoagulant effect via inhibition of thrombin activation,
direct measurement of thrombin activation would be helpful to confirm the anticoagulation
state and secondary thrombus inhibition induced by rivaroxaban (Fig. 5). However, direct measurement of thrombin
activation is impossible because of the short half-life of thrombin. Activated protein C is
another candidate molecule to verify the inhibition of secondary thrombus formation.
Although protein C production is preserved under treatment with rivaroxaban, activation of
protein C is attenuated by a decrease in thrombin–thrombomodulin complex caused by
rivaroxaban-induced inhibition of thrombin formation. However, we decided to measure the
blood levels of TAT as a coagulation marker because a previous study reported that
rivaroxaban inhibited TAT in a dose-dependent manner.21
In the present study, the blood TAT levels were not significantly different between the two
groups and were within the normal range (<3 ng/ml) in both groups. A plausible reason for
this finding is that the tiny emboli and focal thrombotic reaction in this experiment were
insufficient to influence the levels of the coagulation marker, which were measured in
systemic circulating blood samples. However, in clinical settings, it is not necessary to
monitor the blood levels of coagulation markers in patients treated with DOACs. Moreover, it
has been shown that a fixed dose of DOACs is effective as prophylaxis against cardiogenic
embolic stroke in patients with NVAF.12 In
the present study, the plasma concentration of rivaroxaban was in the adequate range in the
rivaroxaban group; therefore, we considered the anticoagulant effect of rivaroxaban as
guaranteed, even in the absence of monitoring of coagulation markers.
To consider the prothrombolytic effect of anticoagulants, the measurement of TAFI
activation is important. TAFI is a plasma procarboxypeptidase that can be activated by
thrombin and the thrombomodulin complex, and activated TAFI inhibits thrombolysis (Fig. 5A).22 A previous report suggested that rivaroxaban reduces thrombin
generation by inhibiting factor Χa, which potentially results in reduced TAFI activation and
leads to thrombolysis (Fig. 5B).5 However, because no assay for TAFI is currently
available in domestic companies, we could not verify this mechanism in our model. Instead,
we measured two markers of thrombolysis, PIC and D-dimers, in the blood. However, the levels
of both markers were below the limit of detection in both groups, and we could not verify
the mechanisms underlying the prothrombolytic effect of rivaroxaban.
Taken together, the results of our study suggest that early initiation of DOACs may improve
the microcirculation in the cerebral cortex by reducing the size of fibrin emboli. Although
a prothrombolytic effect of rivaroxaban was not verified in our study, a relatively dominant
endogenous thrombolytic effect, which resulted in the inhibition of secondary thrombus
formation by exerting an anticoagulant effect, was considered to be the likely mechanism of
the results obtained in our study. A study to compare factor Xa inhibitors, such as
rivaroxaban, and direct thrombin inhibitors, such as dabigatran, would be another way to
investigate the mechanism of embolus size reduction. However, administration of dabigatran
to animals is currently impossible because the drug remains stable only within capsules.
There were three important limitations to our study. First, to consider the clinical
application of our results to stroke therapy, it would have been more rational to administer
rivaroxaban after the injection of the fibrin emboli. However, in our study protocol,
rivaroxaban was designed to be administered prior to the injection of the embolic mixture to
obtain the adequate range of the drug and because of the practical difficulty of
administering rivaroxaban to animals. However, because the fibrin-rich embolic mixture was
prepared ex vivo and did not contain any material from the animal treated
with rivaroxaban, prior administration of rivaroxaban to animals would not be expected to
have any significant effect on the fibrin emboli observed immediately after injection of the
embolic mixture. Furthermore, we must consider an important difference between the clinical
situation and our animal study: in the clinical situation in which drugs are administrated
after the onset of stroke, time is required to obtain an adequate concentration of drugs.
However, in our study, the concentration of rivaroxaban was already in the adequate range
when the embolic mixture was injected. Second, because we used rhodamine dextran to enable
observation of the microemboli, exposure to light and laser radiation could have reduced the
intensity of fluorescence and caused a resultant apparent decrease in the embolus sizes as
observed through the fluorescence microscope. To resolve this problem, we attempted to avoid
unnecessary exposure to light as much as possible. Third, we homogenized the fibrin and used
a filter to exclude emboli that were >40 μm in diameter, although the emboli were not
uniform in size. This could have influenced the distribution of the embolus sizes in the two
groups of mice. Nevertheless, we found no significant difference between the two groups in
the embolus size immediately after injection, which suggests the absence of any significant
effect of variation in the sizes of the emboli on the results of comparison between the two
groups. Furthermore, our model is not suitable for comparison of infarct sizes because the
size of the injected emboli used in this model was too small to cause infarction.
Conclusion
In the current study, we investigated the effect of rivaroxaban on fibrin emboli in
cerebrocortical microvessels by direct observation through the creation of a cranial window
in a mouse model of embolic stroke. The rate of reduction in embolus size was significantly
higher in the rivaroxaban group than in the control group. This result suggests that, rather
than acting as prophylaxis against embolic stroke, the administration of rivaroxaban, even
after embolic stroke has occurred, might be effective in inducing a reduction of embolus
size in the intracranial arteries. Although the underlying mechanism remains unclear, we
believe that early initiation of DOACs in embolic stroke patients might lead to earlier
lysis of emboli relative to that in patients not receiving DOACs. Additional basic and
clinical research is required to determine whether early initiation of DOACs leads to a
better clinical outcome.
Acknowledgements
The authors would like to thank Stefan Heitmeier and Elisabeth Perzborn (Bayer Pharma AG,
Wuppertal, Germany) for sharing their unpublished data, which was used for some of the
interpretations in this article. This work was partially supported by a research fund from
Bayer Yakuhin, Ltd. The funders had no role in the study design, data collection, data
analysis, or preparation of manuscript.
Supplementary video legend
The supplementary video shows the rhodamine-labeled fibrin emboli visualized through the
cranial window. A fibrin-rich embolic mixture was injected into the left common carotid artery, which resulted in stagnation of
the fibrin thrombi within the intracranial arteries on the brain surface.
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