(Stroke. 1995;26:111-116.)
© 1995 American Heart Association, Inc.
Articles |
Acadesine Reduces Indium-Labeled Platelet Deposition After Photothrombosis of the Common Carotid Artery in Rats
From the Departments of Neurology (W.D.D., R.P., N.A.) and Radiology (S.D., M.K.D.), University of Miami (Fla) School of Medicine, and Gensia Pharmaceuticals, Inc (L.P.M., H.G.), San Diego, Calif.
Correspondence to W. Dalton Dietrich, PhD, Department of Neurology (D4-5), University of Miami, School of Medicine, PO Box 016960, Miami, FL 33101.
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Abstract |
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Background and Purpose The adenosine-regulating agent acadesine has been shown to reduce the incidence of myocardial infarction and stroke after cardiopulmonary bypass surgery. The present study examined the effect of acadesine on the accumulation of indium-labeled platelet emboli and infarct size after photothrombosis of the common carotid artery.
Methods Rats were anesthetized with halothane and preloaded with 111In-tropolone–labeled platelets (50 to 80 µCi) 30 minutes before nonocclusive common carotid artery thrombosis induced by a rose bengal–mediated photochemical insult. Intravenous infusion of acadesine (0.5, 1, or 2 mg/kg per minute) or vehicle was begun 30 minutes before right common carotid artery thrombosis and continued for an additional 15 minutes. Rats were then killed and brains processed for the autoradiographic quantitation of labeled platelet aggregates. In a separate group of rats, infarct areas and volumes were determined in treated (acadesine 1 mg/kg per minute) (n=9) and nontreated (n=9) rats 7 days after thrombosis.
Results Although the ratio of right-to-left common carotid artery radioactivity was not affected by treatment, acadesine at 1 and 2 mg/kg per minute significantly decreased (P<.01) platelet deposition within the right cerebral cortex, hippocampus, and striatum. For example, within the frontoparietal cortex, numbers of platelet aggregates were 11.8±1.8 (mean±SEM), 6.1±1.4, 2.3±0.6, and 3.2±0.8 in rats infused with vehicle, 0.5, 1, and 2 mg/kg per minute acadesine, respectively. In addition, infarct volume was reduced by 48% in acadesine-treated (1 mg/kg per minute) rats, with a significant reduction in infarct area at the coronal level 3.7 mm anterior to bregma (P<.01).
Conclusions These results support a prophylactic role for acadesine in reducing the accumulation of platelet emboli during vascular thrombosis and subsequent brain infarction. Acadesine treatment in patients at risk for embolic stroke could potentially lead to cerebral protection.
Key Words: adenosine • embolism • neuroprotection • thrombosis • rats
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Introduction |
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Platelet embolization to the brain is a common consequence of cardiopulmonary bypass surgery as well as cerebrovascular and cardiovascular thrombosis.1 2 In regard to stroke, embolic infarction is the most common form of a clinical stroke.2 Thus, any pharmacological approach that lessens the extent of embolization or hastens its resolution once formed could potentially provide therapeutic benefit in clinical settings in which patients are at risk for stroke.3 4
Adenosine-regulating agents are a class of compounds designed to augment extracellular adenosine levels at sites of tissue injury in an event- and site-specific manner.5 6 7 8 9 10 Studies have shown that the prototypical adenosine-regulating agent acadesine (5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide) potentiates local endogenous adenosine levels during myocardial ischemia.6 In a recent US phase III clinical trial, the use of acadesine in coronary artery bypass surgery significantly reduced the incidence of stroke by 88% (placebo, 4.2%; acadesine, 0.5%; P=.02) and the incidence of combined adverse cardiovascular outcomes by 64% (placebo, 14%; acadesine, 5%; P=.002).11
Recently, morphological and autoradiographic studies have provided experimental evidence for acute platelet embolization into the brain after photochemically induced common carotid artery (CCA) thrombosis.12 13 14 15 16 17 In one investigation, ultrastructural analysis demonstrated non–fibrin-stabilized platelet aggregates as early as 15 minutes after nonocclusive CCA thrombosis.15 In an autoradiographic study that used 111In-labeled platelets, the widespread accumulation of labeled platelets was also documented at 15 minutes after CCA thrombosis.16 Because of the pronounced effect of acadesine in reducing the incidence of clinical stroke associated with coronary artery bypass surgery,11 the present study was designed to examine its application in this thromboembolic animal model of stroke.
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Materials and Methods |
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Animal Preparation
Male Wistar rats weighing 250 to 300 g were anesthetized with 4% halothane and maintained on a 70:30 mixture of nitrous oxide/oxygen and 0.5% halothane delivered via a polyethylene endotracheal tube (PE-240). Femoral arterial and venous catheters (PE-50) were inserted for fluid administration, blood gas determination, and blood pressure monitoring. The rats were mechanically ventilated with a small animal respirator after muscle paralysis with pancuronium bromide 0.35 mg/kg IV, followed by 0.1 mg/kg every 30 minutes for maintenance. Body temperature was maintained at 37°C with a CMA/150 animal temperature controller. With the animal in the supine position the right CCA was exposed under a Zeiss operating microscope, and the beam of a tunable argon/dye laser (562 nm; peak power, 325 mW) was focused onto the CCA.15 18 The methods for platelet labeling with 111In-tropolone have previously been described.16 19 The 111In-labeled platelets were next injected and allowed to circulate for 30 minutes before vascular injury. Intravenous infusion of acadesine at 0.5 (n=10), 1 (n=10), or 2 (n=10) mg/kg per minute was begun 30 minutes before CCA thrombosis and continued for an additional 15 minutes. Nontreated thrombosed rats (n=10) were infused with an equivalent volume of saline. Before irradiation, the photosensitizing dye rose bengal (15 mg/mL in 0.9% saline) was injected at a rate of 10 mg/kg per minute IV for 2 minutes, and the CCA was irradiated for 10 minutes, a procedure that has previously been shown to result in a 50% to 75% stenosis of the irradiated carotid segment.20 Blood samples were obtained for the determination of acadesine plasma levels. Rats were heparinized to prevent postmortem clot formation and were killed with an overdose of potassium chloride injected into the femoral artery at 15 minutes after CCA thrombosis.
Autoradiographic Visualization of 111In-Labeled
Platelets
Surfaces of carotid artery segments were first sponged to remove
loosely adherent blood. Five-millimeter CCA segments were dissected
from the right (irradiated) and left (contralateral) arterial beds and
placed in gamma counter tubes. Thus, right/left (R/L) radioactivity
ratios, normalized to carotid weights, were determined in treated and
nontreated animals.
For the autoradiographic visualization of platelets, brains were removed from the skull and frozen in liquid nitrogen. Frozen sections 10 µm thick were cut in a cryostat and exposed to Kodak SB-5 x-ray film for 5 days. With a dissecting microscope, the regional frequency of labeled platelet foci was determined by an investigator who was blinded to the experimental protocol. In this way, the actual positions of platelet aggregates were noted on line drawings of corresponding coronal brain sections. Regional densities of emboli were determined in the ipsilateral frontal, frontoparietal, and parieto-occipital cortices, striatum, hippocampus, thalamus, cerebellum, and brain stem. For each rat, five brain levels corresponding to 3.7 and 1.7 mm anterior and 1.8, 5.8, and 6.8 mm posterior to bregma were analyzed.21
Infarct Volumes
In a separate series of animals, the effect of acadesine on
infarct volume was determined. Treated rats (n=9) were infused with 1
mg/kg per minute acadesine before thrombosis, and nontreated rats (n=9)
were infused with isotonic saline before, during, and after irradiation
(total infusion period, 45 minutes). At 7 days after thrombosis, rats
were perfusion fixed for histopathologic analysis. At this time the
rats were deeply anesthetized and perfused transcardially with
physiological saline (5 minutes) and then with FAM (a mixture of 40%
formaldehyde, glacial acetic acid, and methanol; 1:1:8 by volume) for
20 minutes at a pressure of 120 mm Hg. The head was immersed in FAM
for at least 24 hours; the brain was then removed and kept in the same
fixative until brains were cut and embedded in paraffin. Brain sections
10 µm thick were prepared at 50-µm intervals and were stained with
hematoxylin and eosin. For morphological study, 10 coronal sections
were selected at defined anatomic levels.22 Each section
was viewed at low power (x10), and the cortical and subcortical
infarcts were traced onto paper by means of a camera lucida microscopic
attachment. Each drawing was then retraced onto a digitizing table
interfaced to a computer, which calculated infarct areas at each
coronal area level. Infarct volume was calculated by numerical
integration of sequential infarct areas.
Statistical Analysis
Physiological data, radioactivity ratios, and emboli counts were
compared by one- or two-way ANOVA, and statistical significance was
assessed by multiple comparison procedures.
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Results |
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Physiological data, including PCO2, PO2, and pH from treated and nontreated thrombosed rats, were within normal ranges. No significant differences were documented between individual animal groups. Blood pressure values were normal, and arterial hypotension was not observed during the study period. Fig 1
summarizes plasma acadesine levels under
the three infusion rates.
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Radioactivity Ratios
The R/L radioactivity ratios of carotid artery segments obtained
from the nontreated and treated groups are shown in Fig 2. All animals demonstrated elevated radioactivity
within the thrombosed segment of the carotid artery. However, no
significant effect of acadesine infusion on R/L radioactivity ratios
was documented. In addition, no significant effect of acadesine
treatment on residual levels of radioactivity within the left
nonirradiated carotid arteries was detected.
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Autoradiographic Findings
Representative autoradiographic images from nontreated
and treated rats illustrating patterns of platelet foci are
presented in Fig 3. In nontreated thrombosed rats,
multiple foci of 111In-labeled platelets were present
throughout the ipsilateral hemisphere. Focal regions of dense
radioactivity appeared overlying the pial surface as well as brain
parenchyma. Bilateral accumulation of platelet emboli commonly appeared
at the level of the frontal cortex. In the more posterior brain
sections, platelet emboli were primarily restricted to the ipsilateral
hemisphere. Emboli were not commonly detected within the cerebellum or
brain stem. In thrombosed rats pretreated with acadesine, the overall
regional density of platelet emboli appeared to be reduced. Reductions
were observed in both cortical and subcortical structures.
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Fig 4 summarizes the regional counts of platelet
aggregates from nontreated and treated animals. Significant effects of
acadesine infusion were seen in frontoparietal and parietal cortices as
well as in striatum and hippocampus. Within the frontoparietal and
parietal cortices and hippocampus, all three infusion rates of
acadesine significantly reduced the number of platelet emboli. In the
striatum, the 1- and 2-mg/kg infusion rate had a significant effect.
Nonsignificant reductions in number of platelet emboli were documented
in the frontal cortex and thalamus with acadesine infusion.
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Histopathologic Findings
At 7 days after CCA thrombosis, small infarcts were detected in
cortical and subcortical regions. The infarcts were well demarcated and
associated with inflammatory cells, including macrophages and reactive
astrocytes (Fig 5). Cortical infarcts were most
frequently detected within border zones (watershed areas) between the
anterior and middle cerebral artery territories and striatum. Infarct
areas were largest at the level of the anterior striatum (Fig 6). In nontreated rats, total infarct volume was
2.26±0.69 mm3 (mean±SEM). Infusion of acadesine at 1
mg/kg per minute decreased infarct volume by approximately 48%
compared with nontreated thrombosed rats (1.09±0.24 mm3).
Because of the rather large range of infarct volumes in both the
treated and nontreated groups, this difference did not reach
significance (P=.11). Although there was a tendency in many
of the regions examined, infarct areas were only significantly
different at coronal level 3.7 mm anterior to bregma (Fig 6).
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Discussion |
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These results demonstrate that nonocclusive CCA thrombosis leads to platelet embolization to the brain and that pretreatment with the adenosine regulating agent acadesine reduces the degree of platelet accumulation in a dose-dependent manner. Previous studies that used this photothrombotic model of CCA thrombosis have indicated that the brain platelets are embolic in origin.12 13 14 15 16 17 Histopathologic studies first demonstrated small brain infarcts after CCA thrombosis,12 13 and more recently, ultrastructural studies have described the morphology of the platelet aggregates to be similar in composition to the carotid thrombus.15 17 In addition, platelet accumulation occurs without severe endothelial damage within downstream microvascular beds or evidence for local platelet aggregation or adhesion.15 17 Thus, while the pathophysiology of brain injury after CCA thrombosis may be multifactorial, platelet embolization is an important component of this injury model.
Recent in vitro and in vivo studies have documented the ability of acadesine to inhibit platelet aggregation. Incubation of human whole blood with acadesine for 120 minutes inhibited ADP-induced platelet aggregation, with an IC50 of 240 µmol/L.23 Administration of acadesine (100 mg/kg IV) to healthy human volunteers resulted in a significant inhibition of ex vivo platelet aggregation induced by either ADP or collagen.23 In a model of unstable angina, acadesine was examined for its ability to attenuate the frequency of recurrent plugging within a stenosed dog coronary artery. In the absence of any hemodynamic effects, acadesine dose-dependently (0.1 to 0.5 mg/kg per minute) reduced the frequency of platelet plugging as monitored by blood flow through the left circumflex coronary artery.24 Acadesine has also been shown to be of value in attenuating reocclusion after coronary thrombolysis in dogs.25 Thus, these studies suggest an antiaggregatory effect of acadesine in a thromboembolic setting.
Because rats were treated with acadesine before CCA thrombosis, the possibility exists that decreased platelet accumulation in the brain was a consequence of the drug affecting the degree of photochemically induced carotid thrombosis. To address this question, the relative amount of carotid thrombus was determined in treated and nontreated rats by comparing R/L carotid radioactivity ratios. In this series, no differences in R/L carotid ratios were detected between the treated and nontreated rats at 15 minutes after irradiation. Thus, the dose-dependent effect of acadesine in reducing platelet accumulation in the brain does not appear to be a consequence of decreasing the magnitude of the carotid thrombus. However, since only a single time period was investigated, we cannot rule out an effect of the drug on the initial size of the nonocclusive carotid thrombus. It also remains to be determined whether acadesine pretreatment affects the subsequent generation of platelet emboli or promotes the disaggregation of emboli.
Acadesine is defined as an event- and site-specific agent that acts by increasing extracellular endogenous adenosine levels in injured tissue.5 6 For example, in a model of myocardial ischemia, acadesine increased adenosine concentrations in blood draining from the ischemic region of the heart.6 Adenosine is a powerful vasodilator,26 27 and elevated adenosine levels have been reported to affect coronary blood flow.10 In the brain, ischemia-induced elevations in the extracellular levels of adenosine have been reported.28 29 Previous hemodynamic studies that used this thrombotic model have documented focal regions of severe ischemia associated with platelet emboli.14 Thus, it is conceivable that in this study, acadesine treatment augmented adenosine release at sites of focal ischemia, leading to pronounced vasodilation and vascular recanalization. In this regard, cerebral blood flow studies would be useful in determining whether acadesine alters the acute hemodynamic consequences of CCA thrombosis.
Recent autoradiographic studies that used this thrombotic model in conjunction with 111In-labeled platelets have documented a high incidence of spontaneous recanalization of brain microvessels after platelet embolization.16 Although platelet accumulation was widespread at 15 minutes after CCA thrombosis, a significant decrease in labeled platelets was documented at 3 hours after thrombosis. Recent ultrastructural studies have also documented that the degree of parenchymal damage seen at sites of occlusive platelet emboli is highly variable and frequently not associated with frank infarction.15 Thus, in this thromboembolic model, therapeutic strategies that promote early reperfusion after platelet embolization should reduce ischemic damage.
In this study acadesine pretreatment significantly decreased infarct area at one coronal level, with a strong tendency in several other regions. Although total infarct volume in treated animals was reduced by 50% compared with controls, these results did not achieve significance. It is not known whether larger study groups, a higher acadesine dose, or longer infusion times would have led to significant differences.
Platelet embolization to the brain is a potential consequence of vascular injury and cardiopulmonary bypass surgery.1 2 Based on clinical and experimental data, the use of adenosine-regulating agents such as acadesine could be advantageous in reducing embolic stroke in some clinical conditions. Whether these agents would provide better protection than other antiaggregatory agents (eg, aspirin) or enhance brain protection when combined with other drugs remains to be determined. Nevertheless, investigations aimed at clarifying mechanisms by which acadesine pretreatment leads to decreased platelet accumulation in the brain after CCA thrombosis should provide insight into novel strategies of brain protection.
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Acknowledgments |
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This study was supported by US Public Health Service grants NS-27127, NS-05820, NS-23244, and HL-47201 and a grant from Gensia, Inc. The authors appreciate the technical support of Dr Brant Watson, Dr Abdol K. Ghafouripour, Mansoor Kapadvanjwala, and Christina Pavia.
Received May 18, 1994; revision received September 7, 1994; accepted September 20, 1994.
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