Published online before print February 21, 2008, doi: 10.1161/STROKEAHA.107.496448
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(Stroke. 2008;39:1262.)
© 2008 American Heart Association, Inc.
Original Contributions |
Blocking of Platelets or Intrinsic Coagulation Pathway–Driven Thrombosis Does Not Prevent Cerebral Infarctions Induced by Photothrombosis
From the Departments of Neurology (C.K., S.B., M.A., G.S.), Neuroradiology (M.P., I.N., M.B.), Clinical Biochemistry and Pathophysiology (T.R., B.N.), and the Rudolf Virchow Center for Experimental Biomedicine (B.N.), University of Würzburg, Germany.
Correspondence to Guido Stoll, MD, Department of Neurology, University of Würzburg, Josef-Schneider-Str 11, D-97080 Würzburg, Germany. E-mail stoll_g{at}klinik.uni-wuerzburg.de
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Abstract |
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Background and Purpose— Models of photochemically-induced thrombosis are widely used in cerebrovascular research. Photothrombotic brain infarctions can be induced by systemic application of photosensitizing dyes followed by focal illumination of the cerebral cortex. Although the ensuing activation of platelets is well established, their contribution for thrombosis and tissue damage has not formally been proved.
Methods— Infarction to the cerebral cortex was induced in mice by Rose Bengal and a cold light source. To assess the functional role of platelets, animals were platelet-depleted by anti-GPIb
antibodies or treated with GPIIb/IIIa-blocking F(ab)2 fragments. The significance of the plasmatic coagulation cascade was determined by using blood coagulation factor XII (FXII)-deficient mice or heparin. Infarct development and infarct volumes were determined by serial MRI and conventional and electron microscopy.
Results— There was no difference in development and final size of photothrombotic infarctions in mice with impaired platelet function. Moreover, deficiency of FXII, which initiates the intrinsic pathway of coagulation and is essential for thrombus formation, or blockade of FXa, the key protease during the waterfall cascade of plasmatic coagulation, by heparin likewise did not affect lesion development.
Conclusions— Our data demonstrate that platelet activation, factor XII–driven thrombus formation, and plasmatic coagulation pathways downstream of FX are not a prerequisite for ensuing tissue damage in models of photothrombotic vessel injury indicating that other pathomechanisms are involved. We suggest that this widely used model does not depend on platelet- or plasmatic coagulation-derived thrombosis.
Key Words: coagulation factor XII • GPIb • GPIIb/IIIa • photothrombosis • platelets
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Introduction |
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Photochemically-induced vascular thrombosis is widely used to mimic thrombotic disorders in various organ systems, eg, the spinal cord, carotid artery, gut, and inner ear because of its convenience and reproducibility.1–9 In 1985, Watson et al1 introduced brain photothrombosis as a simple model by which cortical infarcts can be induced in rats. To achieve thrombosis a photosensitive dye is injected systemically into the circulation and external focal illumination through intact tissue is performed. This leads to local activation of the dye with subsequent free radical formation and photoperoxidation of the endothelium.2,10 Endothelial injury is then thought to mediate clot formation in illuminated vessels. Accordingly, platelet aggregation11,12 and platelet-containing thrombi have been demonstrated in many different settings of photothrombosis (PT) by electron microscopy.2,4,8 The key question, however, whether platelet activation and thrombus formation is mandatory for ensuing tissue damage, has not yet been systematically addressed. We pretreated mice with antibodies against platelet receptors leading to complete platelet depletion (anti-GPIb
) or their functional inhibition (anti-GPIIb/IIIa). In addition, we induced PT in mice lacking coagulation factor XII (FXII), an indispensable factor for pathological thrombus formation.13,14 In another subgroup, FX activity was blocked by low-molecular-weight heparin. Surprisingly, serial MRI and histopathology revealed that cerebral photothrombotic infarctions developed to the same extent in the absence of functional platelets or FXII or FX activity. |
Materials and Methods |
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Antiplatelet Antibodies
The monoclonal antimouse GPIIb/IIIa antibody JON/A was produced and characterized as described.15 F(ab)2 fragments from JON/A were generated by 12-hour incubation of 10 mg/mL antibody with immobilized pepsin (Pierce), and the preparation was then applied to an immobilized protein A column followed by an immobilized protein G column (Pharmacia) to remove Fc fragments and any undigested IgG. The purity of the F(ab)2 fragments was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of the gel. F(ab)2 fragments of JON/A completely inhibit aggregation of mouse platelets.15 Antimouse GPIb
antibodies were purchased from Emfret Analytics. Antibodies against GPIb induce profound and irreversible thrombocytopenia in mice.16 For control experiments, purified rat IgG2 (Serotoc) was applied.
Platelet Preparation and Counting
Mice were bled under ether anesthesia from the retroorbital plexus. Blood was collected in a tube containing 10% (v/v) 0.1 mol/L sodium citrate or 7.5 U/mL heparin, and platelet rich plasma was obtained by centrifugation at 300g for 10 minutes at room temperature (RT). For determination of platelet counts, blood (20 µL) was obtained from the retroorbital plexus of anesthetized mice using siliconized microcapillaries and immediately diluted 1:100 in Unopette kits (Becton Dickinson). The diluted blood sample was allowed to settle for 20 minutes in an Improved Neubauer hemocytometer (Superior), and platelets were counted under a phase contrast microscope at x400 magnification.
Bleeding Time Experiments
Mice were anesthetized by intraperitoneal injection of 150 µL of a mixture of 0.08% xylazine base (Rompun) and 1.6% ketamine (Imalgen 1000), and 3 mm of the tail tip was amputated with a scalpel. The tail was then blotted with filter paper every 15 seconds until the paper was no longer blood-stained. Where necessary, bleeding was manually stopped at the 20-minute time point to prevent death.
Animal Studies
All animal experiments were approved by the Regierung von Unterfranken and conducted according to the recently published recommendations for research in mechanism-driven basic stroke studies.17 Focal cerebral ischemia was photochemically induced in male mice weighing 20 to 25 g as described.18 In brief, mice were placed into a stereotactic device. A cold light source was positioned 2 mm posterior and 2.4 mm lateral to the Bregma. Then 0.2 mL of Rose Bengal solution (Sigma; 10 g/L in normal saline) were administered intraperitoneally, and the brain was illuminated through the intact skull for 20 minutes. Animals were kept in inhalation anesthesia (2.5% enflurane in a 70% N20 / 30% O2 mixture) and normothermia throughout surgery. A total of 18 C57/Bl6 mice pretreated with anti-GPIb antibodies, 9 pretreated with JON/A F(ab)2, 7 pretreated with low-molecular-weight heparin (fraxiparin, 200 U/kg), 7 pretreated with low-molecular-weight heparin (fraxiparin, 200 U/kg) plus anti-GPIb antibodies, and 20 control mice receiving purified rat IgG2 (Serotec) were subjected to PT. Antibodies were injected either intraperitoneally 24 hours (anti-GPIb) or intravenously 1 hour (JON/A) before the start of the experiment, whereas heparin was applied immediately before the induction of PT. In addition, we induced PT in 5 FXII KO mice13,14 and 6 matched WT controls.
MRI
MRI measurements were performed using a clinical 1.5 Tesla MRI unit (Magnetom Vision; Siemens). Serial MRI studies were obtained from animals which had received antiplatelet antibodies or from controls at 4 hours, 24 hours, 3 days, and 7 days after PT. FXII KO and WT animals were examined at day 1.
During MRI scans, anesthetized animals were lying in a supine position with their heads fixed into a custom-made dual-channel surface coil designed for investigations of the mouse brain (A063HACG; Rapid Biomedical). The MR protocol included a scout sequence in 3 planes, a coronal T2-w sequence (TR 2.500 ms, TE 92 ms) with a slice thickness of 2 mm, and a coronal CISS sequence (TR: 16,4 ms, TE: 8,2 ms) with a slice thickness of 1 mm. For postprocessing, MR data were transferred to an external workstation (Leonardo; Siemens). Infarct volumes for each treatment group and time point were calculated as described elsewhere19 and normalized to the total volume of the ipsilateral hemisphere.
Light Microscopy
At least 3 animals per group were euthanized 24 hours after photochemically-induced cortical infarction. Brains were rapidly removed, immersed in 4% paraformaldehyde (PFA) for fixation, and embedded in paraffin. Multiple 5-µm thick coronal sections were cut at the level of the infarction, which can be identified as a round pale area on the brain surface. For evaluation of tissue damage sections were hematoxylin-eosin (H&E)-stained and viewed using a Zeiss Axiophot microscope (Zeiss).
Transmission Electron Microscopy
For transmission electron microscopy, 3 control animals were transcardially perfused with 4% PFA/2% glutaraldehyde at 5 minutes after PT. Brains were removed and fixed overnight in 4% PFA/2% glutaraldehyde. After washing in cacodylate buffer and osmification, the tissue was acetone-dehydrated and embedded in spurr. The region of interest was identified in methylene blue–stained 500-nm-thick sections. Then, 80-nm-thick sections were cut, mounted on grids, contrast-enhanced using lead citrate, and visualized under an electron microscope (EM 10C/CR; Zeiss).
Statistical Analysis
Statistical analysis was performed using the Prism 4 software package (GraphPad Software). Data are expressed as mean±SD. To analyze the development of lesion volumes over time, nonparametric Kruskal-Wallis tests followed by Dunn posthoc multiple comparison tests were applied. The infarct size in FXII KO and WT animals was compared using an unpaired, 2-tailed Student t test. Probability values <0.05 were considered statistically significant.
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Results |
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To assess the role of platelets in PT lesion formation, we used antibodies to either render mice severely thrombocytopenic or to completely inhibit platelet aggregation/thrombus formation in vivo. For platelet depletion, mice received 100 µg of a mixture of anti-GPIb
antibodies intraperitoneally 24 hours before challenge. This treatment has been shown to deplete >95% of the circulating platelets in mice within minutes by Fc-independent mechanims and to maintain the platelet count below 0.05x106/µL (normal: 
1x106/µL) for at least 3 days (Table 1).16 In agreement with these reports, platelet counts were reduced by >98% in anti-GPIb treated mice as compared to control mice 24 hours after antibody injection. Because of this severe thrombocytopenia, tail bleeding times were consistently greater than 20 minutes in those animals. In contrast, control mice arrested bleeding within 4.8±3.8 minutes (Table 1).
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As demonstrated before in rats,19,20 photothrombotic infarctions in mice appeared as hyperintense cortical lesion on T2-w MRI sequences (Figure 1A). All lesions significantly increased in size from 4 to 24 hours after PT indicating secondary infarct growth. Thereafter infarct volumes gradually decreased. Unexpectedly, depletion of platelets by GPIb antibodies did not affect infarct size and the kinetics of infarct development as revealed on T2-w images by MRI in comparison to controls (Figure 1B and 1C; Table 2).
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A second group of mice received 100 µg F(ab)2 fragments of the function blocking anti-GPIIb/IIIa antibody, JON/A, intravenously 1 hour before challenge. In agreement with previous reports,21 this treatment had no significant effect on peripheral platelet counts but completely inhibited ex vivo platelet aggregation in response to different stimuli, and consequently resulted in tail bleeding times consistently greater than 20 minutes (Table 1). Again, photothrombotic brain lesions were not different between groups (Figure 1C; Table 2). Light microscopical examination of H&E-stained sections at 24 hours after photochemical induction of cortical infarction did not reveal differences in lesion development between mice treated with antiplatelet antibodies, and control animals, thus confirming our MRI findings (Figure 2). The hyperintense cortical lesions on MRI corresponded to infarcted brain tissue in all experimental groups. However, hemorrhagic transformation of PT lesions was seen in anti-GPIb–treated thrombocytopenic mice (Figure 2B; Table 2) and to a lesser extent in mice after GPIIb/IIIa blockade (Figure 2C; Table 2), but not in FXII–/– or untreated mice (Figure 2A and 2D; Table 2). In line with previous studies,1,18 infarcts were sharply demarcated from the surrounding normal brain tissue. By transmission electron microscopy we could demonstrate prominent red blood cell stasis as well as intravascular platelet aggregates in control mice after PT (Figure 3A), thus confirming earlier observations by Dietrich et al.2 These data show that intravascular thrombi are formed early in this model, but based on the lacking effect of antiplatelet treatment this appears to be a secondary phenomenon.
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The surprising finding that cerebral PT lesions developed independently of platelet aggregate formation strongly suggested that this process may not be related to thrombotic activity in the injured area of the brain. To further substantiate this finding, we used mice lacking coagulation factor XII (FXII). These mice have previously been shown to be profoundly protected from pathological thrombus formation in injured arteries13 as well as after transient MCAO,14 which appears to be based on a marked inability of the animals to generate sufficient amounts of thrombin at sites of injury to stabilize the platelet-rich thrombus. As shown in Figure 3 (B and C) infarct size and the kinetics of infarct development were indistinguishable between control and FXII–/– mice (see also Table 2).
The extrinsic and intrinsic branch of the plasmatic coagulation cascade converges in the activation of FX. To test whether FX activity is essential for infarct development, mice received 200 U fraxiparin/kg immediately before the induction of PT. Again, no differences were observed concerning infarct size, kinetics of infarct development, or histological alterations compared to control animals (Figures 1C and 2
E; Table 2).
To rule out that the remaining alternative clotting mechanism (e. g. platelet or plasmatic coagulation driven thrombosis) which had not been blocked by either monotherapy contributed to in vivo thrombus formation, we also pretreated mice with fraxiparin and anti-GPIb antibodies before the induction of PT. This combined antithrombotic treatment induced intracerebral hemorrhage in 100% of the animals (7 of 7 mice until day 3) without obvious clinical signs (Figure 4; Table 2). Intracerebral hemorrhage was detected by MRI as hypointense rim around the hyperintense photothrombotic infarct core (Figure 4A) and confirmed macroscopically on whole brains by autopsy (Figure 4B). Consequently, brain lesion volumes at either time point were significantly higher compared to controls (Figure 4C).
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Discussion |
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As principal finding, our study demonstrates that platelets are not essential for the induction and development of photothrombotic cortical brain infarctions. In vivo blockade of platelet function or depletion of platelets did not alter the infarct size measured by MRI. Supporting this, histological analysis of the cortical infarctions did not reveal differences between the groups.
Platelet adhesion and aggregation at sites of vessel injury is crucial to arrest posttraumatic blood loss but is also considered to play a central role in the onset of acute ischemic cardio- and cerebrovascular events such as myocardial infarction and stroke.22,23 GPIIb/IIIa antagonists are currently considered the most powerful inhibitors of platelet function in prothrombotic events,24,25 as they inhibit the final common pathway of platelet aggregation, irrespective of the agonist that stimulates the cells. This has also been confirmed in mice where F(ab)2 fragments of the blocking anti-GPIIb/IIIa antibody JON/A abrogated platelet aggregation in different experimental thrombosis models providing more or less complete antithrombotic protection.21,26,27 Our finding that JON/A-F(ab)2 failed to reduce the infarct size or to alter the kinetics of infarct development after the induction of PT demonstrates that platelet aggregation is not a prerequisite for lesion development in this model. This is further confirmed by the normal lesion formation in mice treated with anti-GPIb antibodies to reduce their circulating platelets counts to less than 2% of normal. These mice display a severe hemostatic defect and are completely protected in models of arterial thrombosis (Nieswandt et al, unpublished, 2007) showing that platelet mediated processes are not essential for lesion formation in the PT model. However, in untreated control mice we could demonstrate prominent intravascular platelet aggregates after PT by transmission electron microscopy, confirming previous histological observations2,4,8 and recent MRI evidence.20 Interestingly, Dietrich and colleagues2 observed that hypothermia had also a profound effect on clot formation in cerebral PT. Importantly, in spite of reduced platelet aggregation in hypothermic animals, infarct size at 3 days did not differ. Similarly, Rose Bengal–induced thrombosis of mesenteric arterioles did not respond to antiplatelet therapy with acetylsalicytic acid.28
In the absence of functional platelets, occluding thrombi could potentially be formed by the plasmatic coagulation cascade. We could show, however, that targeting FXa, the key protease of plasmatic coagulation by low-molecular-weight heparin likewise had no influence on infarct development in the PT model. This is in line with a previous study in which enoxaparin reduced the formation of brain edema after PT but had no influence on stroke size.29 In addition infarct development in the PT model is independent from FXII. This is in contrast to findings reported for the transient MCAO paradigm. Here FXII KO mice were protected from cerebral ischemia by a dramatic reduction of intravascular fibrin thrombus formation similar to wild-type mice that received the potent FXII inhibitor PCK.14 To rule out that platelet deficiency would be compensated by plasmatic coagulation driven thrombosis and vice versa we finally used a combined treatment strategy. The complete disruption of pathways of thrombus formation by platelet depletion and administration of heparin again did not reduce infarct volumes but increased the frequency of intracerebral bleeding after PT.
Our data show that platelet aggregation and thrombus formation, although occurring in models of PT, appear not to be essential for lesion development in the brain. In the presence of antiplatelet drugs and during anticoagulation alternative pathways must be operative to induce neuronal and glial cell death. Cerebral PT is characterized by rapid breakdown of the blood brain barrier which is accompanied by strong parenchymal edema.2,10 Therefore, edema-induced secondary vascular compression could also critically account for lesion development during PT. Other pathomechanisms could involve cytotoxic radicals and secondary inflammatory cascades, eg, activation of microglia and release of cytokines.18,19,30,31
Taken together, the fact that ischemia occurs to the same extent in mice with platelet and coagulation cascade deficiencies suggests that these factors are not a necessary aspect of stroke from PT. These unexpected findings reveal differences in mechanisms of tissue injury induced by PT as compared to focal ischemia induced by MCAO, where platelet activation and the intrinsic coagulation pathway are instrumental.14,23 In conclusion, when evaluating antithrombotic pharmaceuticals to limit tissue injury using PT models, one should be cautious because ensuing platelet containing thrombosis may be present, but not mandatory to induce tissue damage.
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Addendum |
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After our manuscript had been accepted for publication in Stroke and underwent copyediting, a complementary study was published by Frederix and co-workers that confirmed our present data (Frederix K, Chauhan AK, Kisucka Z, Zhao BQ, Hoff EI, Spronk HM, Ten Cate H, Wagner DD. Platelet adhesion receptors do not modulate infarct volume after a photochemically induced stroke in mice. Brain Res. 2007;1185:239–245.). In line with our pharmacological findings, mice mutant in different crucial platelet adhesion and activation receptors (eg, vWF, GPIB
, β3integrin) did not show any differences in lesion development when subjected to cortical photothrombosis.
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Acknowledgments |
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The expert technical assistance of Gabi Köllner, Tanja Horn and Heinrich Blazyca (Würzburg) is gratefully acknowledged. We thank Igor Kobzar (Würzburg) for help with transmission electron microscopy.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft SFB 688 (grants A1, A3 and B1 to M.B., B.N., T.R., and G.S.). M.B. holds an endowed professorship donated by the Bayer-Schering Pharma AG, Berlin to the University of Würzburg. I.N. was supported by a research grant of the Medical Faculty of the University of Mannheim.
Disclosures
None.
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Footnotes |
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C.K. and S.B. contributed equally to this study.
Received June 13, 2007; revision received July 31, 2007; accepted August 27, 2007.
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References |
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