University of Rochester, Department of Environmental Medicine, Rochester, New York
1 To whom correspondence should be address at Proctor and Gamble Co., FemCare Innovation Center 1B17, 6110 Center Hill Avenue, Cincinnati, OH 45224. E-mail: silvasegarra.vm{at}pg.com.
Received January 6, 2005; accepted March 3, 2005
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ABSTRACT |
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Key Words: ultrafine particles; polystyrene particles; thrombosis; ear vein; rose bengal.
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INTRODUCTION |
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Understanding the mechanism by which UFP interact with the vascular system requires the use of animal models of endothelial injury. In fact, several such models has been developed, including those in which injuries are produced mechanically (Stockmans et al., 1991), electrically (Carmeliet et al., 1997
), chemically with FeCl3 (Farrehi et al., 1998
; Kurz et al., 1990
) and with lasers and photochemicals (Kawasaki et al., 1999
, 2000
; Kochevar et al., 1994
; Nemmar et al., 2002
; Roesken et al., 1998
) in large vessels such as the femoral vein or carotid artery. Of particular interest to us was the Rose Bengal (RB) thrombosis model. This model is based on the local photodynamic generation of singlet molecular oxygen from systemically injected RB that is illuminated with green light (GL, 540 nm) (Umemura et al., 1990
). Peroxidative damage to endothelial membranes provides the initial stimulus for platelet adhesion and eventual formation of thrombi (Kochevar et al., 1994
; Saniabadi et al., 1995
). Evaluation of thrombogenesis in this system requires that the vessel of interest be mounted on a transilluminator, which allows visualization and imaging of the growing thrombus. Factors affecting thrombus formation include plasma RB concentration, time post RB administration, and total exposure time to green light (Kawasaki et al., 1999
; Saniabadi et al., 1995
). Also, manipulation of the vessel itself can affect RB-induced endothelial responses, which can contribute to increased variability of the results when using this invasive technique. Therefore, the major objectives of this article are (1) to adopt a noninvasive model without the disadvantages associated with previous models and (2) to validate this model for evaluating particle-induced thrombogenic effects by confirming the results of Nemmar et al. (2002)
indicating that iv and intratracheal administration of UFP accelerates venous thrombus formation. This article presents a modified version of the RB model in which the rat ear vessel is used rather than exposing and manipulating the femoral vein. A similar technique has been reported by Rosen et al. (2001)
in which the ear vessels of C57BL6 mice were imaged in situ to study differences between the RB- and laser-induced vascular damage. These experiments demonstrated that this noninvasive technique requires minimal handling and preparation of the vessel, thereby reducing unwanted vascular effects. Also, because the local injuries generated are small, there is no major perturbation of the coagulation system and no significant consumption of coagulation factors. Thus, multiple injuries can be induced and recorded in a single animal (Rosen et al., 2001
), which allows for reduced numbers of animals per experiment. In addition, the ear section can be excised, fixed, and prepared for histology and immunohistochemical analyses of local endothelial dysfunction markers such as fibrinogen, endothelin, von Willebrand factor (Muller and Griesmacher, 2000
), and P-selectin (Becker, 2001
). More importantly, as our data show, the ear vein model is useful in studying the thrombogenic effects of particles administered to the lungs.
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METHODS |
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Rose Bengal route of administration.
Animals received either intraperitoneal (ip, 200 mg/kg), iv bolus (10 mg/kg), or iv infused (24 mg/kg/h) doses of RB in different experiments to characterize the kinetics of plasma RB accumulation after each route of administration. For this purpose, blood was collected at different times from the tail vein (125 µl) for up to 90 min after dosing. Briefly, a small incision was made on the tail vein, from which half of a 250 µl heparinized blood collecting tube was filled (VWR International, West Chester, PA). Bleeding was stopped by applying pressure to the vessel. The same site was used for subsequent collections. Blood was then transferred into a 0.250-ml microcentrifuge tube covered with aluminum foil and centrifuged at 10,000 x g for 10 min to obtain plasma. Rose Bengal was quantified in plasma via its absorbance at 549 nm. The goal of these experiments was to select a method of RB administration that achieves and maintains a stable RB plasma level.
Rose Bengal thrombosis model.
Rats were anesthetized with sodium pentobarbital (30 mg/kg, ip) and placed supine on a heating pad (37°C). A depilatory was used to remove hair from the ears. Both femoral veins were exposed and cannulated with a 15-gauge metal catheter with a Silastic tubing tip (0.025 in. inner diameter and 0.47 in. outer diameter). One femoral vein was used for RB administration; the other, for intravenous administration of the ultrafine particles. Animals were then placed on an inverted microscope stage (Olympus 1X51, Olympus America Inc., Melville, NY) such that one ear rested on a glass slide (1-mm-thick) in the optical path so that the blood vessels and flow could be visualized with a digital camera (Spot Camera Software version 4, Spectra Services, Webster, NY). The ear was kept moist with saline throughout the observation period, and body temperature was maintained between 37°C and 38°C with the help of a heating lamp. A 2-mm green laser beam (532 nm, 100 mW, Extreme Lasers, Seabrook, TX) was introduced to the optical system from the back of the microscope through the 10x objective to be focused on a small secondary or tertiary ear vein (7090 µm). To facilitate focusing on the vein without inducing damage to the vessel, a neutral filter was placed between the laser and the microscope objective. Light intensity at this spot was measured with an optical power meter (Model 1815-C, Newport Corporation, Irvine, CA) and was found to be close to 0.0 mW with the filter and between 13 and 14 mW without the filter. At that time, separate sections of the target ear vessel were illuminated (for 60240 s) with the green light (GL, 13.5 mW) alone to determine the laser effect on the vessel, or the baseline value. Rose Bengal was administered 5 to 10 min later, and the vessel was illuminated (for 30120 s) in different sections to determine the effect of RB and GL on blood coagulation. In this way, each animal served as its own control. Then UFP were either administered iv or instilled intratracheally (see below under Aminated and Carboxylated Polystyrene Particles). After the particles were in the system for 2 min, the vessel was illuminated (for 1560 s) repeatedly for 1 h, in different sections of the same vessel or of another vessel of the same size, to study the effects of UFP on thrombus induction. In other experiments, rats did not receive the photosensitive dye (Rose Bengal) prior to particle administration, and local endothelial activation/injury was induced with green laser illumination only.
To determine if the baseline value (effect of GL alone) changes with time, a group of animals received iv saline instead of UFP, and illumination time required to induced thrombus formation was assessed 10, 20, 30, and 60 min thereafter. The same experiment was repeated 2 and 3 days after the initial exposure in the same rat.
Thrombus formation is defined and evaluated as follows: A thrombus should occur within a maximum of 30 s after GL exposure for the determination of green laser illumination time. This illumination time is defined as the thrombus-inducing time (TIT). Reproducibility of TITs was verified in a different spot on the vein. Because TIT in the presence of RB (RB-TIT) is shorter than baseline-TIT, RB-TIT was determined by starting with half of baseline-TIT; if a thrombus was induced, the time was halved again, and so on. Likewise, this principle of halving TIT was also applied after UFP dosing to determine particleinduced-TIT (UFP-TIT). In contrast, if no thrombus was induced, illumination time was doubled until UFP-TIT could be defined.
Aminated and carboxylated polystyrene particles.
To validate the ear vein model, experiments were performed using both ultrafine (60 nm) aminated (positively charged) and carboxylated (negatively charged) polystyrene particles (Bangs Laboratories, Fishers, IN). Particles were sonicated for 15 min and vortexed prior to suspension in saline, as well as before iv and IT administration to rats. To study their effects on coagulation as described above, different doses of aminated (0.02, 0.5, and 50 mg/kg) and carboxylated (0.1 and 50 mg/kg) particles were given in a volume of 250 µl after a steady level of RB was reached by iv infusion. In experiments where RB was not used, 0.5 mg/kg dose of aminated particles was administered iv or IT 10 to 15 min after baseline-TIT was determined.
Data analysis.
Because our baseline-TIT measurements showed large variability between different rats but were reproducible in the same individual animal, we used the ratios of RB-TIT to baseline TIT or UFP-TIT to RB-TIT to assess the effects. Thus, a ratio of 1 would indicate no effect, and a ratio of 0.5 a 50% reduction of illumination time to induce thrombus formation. In experiments where rats did not receive RB prior to particle administration, the effect was calculated as the ratio of UFP-TIT to baseline-TIT. Results were analyzed for statistical differences by one-way analysis of variance (ANOVA). Differences between groups were further analyzed with the Tukey-Kramer multiple comparison test. Such differences were considered significant when p 0.05.
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RESULTS |
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DISCUSSION |
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In vivo experimental models of thrombosis are essential for understanding the mechanisms involved in thrombogenesis. Most of the available models require extensive surgical procedures that may inadvertently cause activation of the coagulation system in the vessel of interest, thus affecting data interpretation. Therefore, the aim of this study was to develop an alternative model that is less invasive and that is independent of RB, and to test its utility in the evaluation of previously reported thrombogenic effects of UFP. We took advantage of a recently published injury model in which damage was induced photochemically by illuminating the ear of C57BL6 mice with a green laser after injection with RB (Rosen et al., 2001). In this model as well as ours, it was possible to focus the laser beam through the objective of an inverted microscope to create the injury on specific sections of the ear vessels.
The architecture of the ear vasculature varies among rats. However, there are generally one or two primary veins proximal to the head with a diameter of approximately 250 to 350 µm. This primary vein is fed by secondary veins of 60 to 200 µm, which in turn are fed by tertiary vessels of 60 to 90 µm. The smaller secondary vessels (7090 µm) were used to induce injury in this investigation to maintain anatomically similar vascular parameters between experiments. The use of microscopy allowed monitoring of thrombogenesis by transillumination in real time (Gavins and Chatterjee, 2004; Rosen et al., 2001
; Stockmans et al., 1991
).
The ear vein technique has several advantages. First, the target vessel is easy to identify and does not have to be isolated via invasive procedures. For instance, our approach only requires that the ear of an anesthetized rat rests flat on a glass slide to be imaged by a digital camera, thus avoiding unwanted manipulation of the vessel. A drop of saline or distilled water is enough to keep the ear flat for long periods of time. According to Rosen et al., 2001, the ear can also be taped to the slide for easy imaging. However, this may potentially induce some pressure on ear vessels, and we did not find this to be necessary. It is also important to mention that because of the noninvasive nature of this technique, there is the additional benefit of better animal care. In addition, because the injuries generated are small, multiple injuries can be induced in both ears of the same animal. We also found that baseline-TIT did not change in vessels of both ears of the same rat, even 2 to 3 days after the first determination. This will result in a reduced number of animals used per experiment. Because fluctuations in RB plasma concentration affect the severity of the endothelial response (Saniabadi et al., 1995
), another advantage of our model is that RB iv infusion produces a steady-state plasma level of the photosensitive dye within 30 min. Our data show that this steady-state concentration of RB (60 µg/ml) produces a constant and reproducible vascular response within the same animal even 60 min after the start of the iv infusion.
To validate our model for its use in the study of UFP-induced thrombosis, we used surface-modified polystyrene particles (PSP) 60 nm in diameter. Although not environmentally relevant, PSP offer the advantage of being well characterized in size, are not cytotoxic (Nemmar et al., 2002), and may represent a model for engineered nanoparticles. The thrombogenic potential of these particles has previously been documented in a peripheral model of experimental thrombosis in hamsters (Nemmar et al., 2002
). In these studies, iv-administered positively charged PSP enhanced thrombus formation by 219% (50 µg/kg) and 307% (500 µg/kg) when compared to saline controls, whereas negatively charged particles at similar doses did not affect thrombus formation. Our results confirm the data of Nemmar et al., because iv administration of different doses of aminated PSP induced dose-dependent significant reductions in RB-TIT, indicating accelerated development of thrombosis in the presence of these UFP. It appears, though, that the dose-dependent thrombogenic effect of positively charged PSP is limited to a certain dose range. At a very high dose of 50 mg/kg this effect was weaker, although still significant (Fig. 3). A likely explanation is that at this high dose, administrated in a small volume of 250 µl, PSP agglomerate quickly to form larger clusters which may not be as effective in inducing thrombi. Indeed, Nemmar et al. (2003a)
have reported that larger 400-nm particles were not thrombogenic in their model.
Thus, our results using the noninvasive ear vein model and those of Nemmar et al. (2002; 2003a
) using the femoral vein model agree quite well with each other with respect to the thrombus-inducing potential of positively charged PSP, as well as with respect to lack of such potential for negatively charged PSP. This agreement between our results and those of Nemmar et al. (2002)
demonstrates the usefulness of our noninvasive model of in vivo thrombus formation.
However, quantification of thrombus formation as we assessed it can still be improved in the ear vein model. As described earlier under Methods, thrombus formation is determined as a fraction of RB-TIT based on an immediate response after green laser illumination; i.e., laser illumination time is changed. In contrast, Nemmar and colleagues measured thrombus development for a longer period (40 min) without changing laser illumination time, and they used computer software to quantify the intensity of the injury. Our laboratory is working on the development of a procedure to quantify the damage in our ear model using a similar computerized image-processing system and keeping the laser illumination time constant.
In an effort to further simplify our model and reduce the number of variables in each experiment, we explored the effect of UFP on thrombus formation after direct laser injury without the use of RB. We observed that illumination time in this system was slightly higher than with the RB model. With this model, rats then received 0.5 mg/kg aminated-PSP iv, and thrombus formation was assessed. It was found that thrombogenesis in the presence of UFP was significantly increased (51% reduction in TIT) after administration of these positively charged PSP (Fig. 4). This response was comparable to the earlier studies in our RB ear model in which aminated-PSP reduced illumination time to 33% of baseline-TIT. Together, these data indicate that the thrombogenic effect of these particles is independent of RB. With respect to the mechanism of thrombus induction by GL alone, Rosen et al. (2001) showed that direct green laser illumination without RB induces significant endothelial injury (highly vacuolated cells) without denuding the vessel, leading to adhesion of numerous platelets and polymorphonuclear leukocytes (PMNs). Our data showing that IT instillation of aminated PSP induces a significant reduction in TIT in much the same way that iv injected PSPs do support the hypothesis that ultrafine particles translocate out of the lung and can directly affect thrombus formation. The time course of response (i.e., thrombus induction very soon after IT administration) adds further support to this hypothesis. If a lag in thrombus induction after IT administration were observed, this would suggest that inflammatory mediators are required for the effect instead of direct contact of the particles with endothelial cells, platelets, and/or circulating inflammatory cells. This has also been suggested by Nemmar et al. (2004)
. We conclude that this simplified version of the rat ear vein model, without the use of RB, has a number of advantages and is a valid alternative to the classical femoral vein model.
Further studies are needed to determine the mechanism of UFP-induced thrombogenesis. Interestingly, an article by Berry et al. (1977) presented the first evidence that UFP can indeed translocate from the lung into the bloodstream and accumulate inside blood platelets. In their studies, IT-administered ultrafine (30 nm) colloidal gold particles were identified inside blood platelets in alveolar capillaries of Wistar rats as soon as 30 min after instillation, indicating that platelets may be important target cells for UFP translocating across the alveolar-capillary barrier. The importance of platelets in thrombogenesis induction by airborne ambient UFP has further been demonstrated by showing that platelet activation time was significantly shortened after adding diesel exhaust particles to untreated hamster blood (Nemmar et al., 2002
). Thus, we hypothesize that once UFP reach platelets they can induce platelet activation, resulting in their aggregation after interaction with injured or activated endothelial cells. Ultrafine particles could, for example, induce expression of CD40 ligand (CD40L), a transmembrane protein expressed in activated platelets and endothelial cells that has been associated with increased risk of cardiovascular events (Vishnevetsky et al., 2004
).
Thus, the objective of our future studies will be to determine the contribution of inhaled environmental UFP in thrombus induction in the noninvasive ear vein model.
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ACKNOWLEDGMENTS |
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