November 1999
Volume 40, Issue 12
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Retina  |   November 1999
In Vivo Evaluation of Platelet–Endothelial Interactions in Retinal Microcirculation of Rats
Author Affiliations
  • Akitaka Tsujikawa
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine;
  • Junichi Kiryu
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine;
  • Atsushi Nonaka
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine;
  • Kenji Yamashiro
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine;
  • Hirokazu Nishiwaki
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine;
  • Shinichiro J. Tojo
    Sumitomo Pharmaceuticals Research Center, Osaka;
  • Yuichiro Ogura
    Department of Ophthalmology, Nagoya City University Medical School, Japan.
  • Yoshihito Honda
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine;
Investigative Ophthalmology & Visual Science November 1999, Vol.40, 2918-2924. doi:
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      Akitaka Tsujikawa, Junichi Kiryu, Atsushi Nonaka, Kenji Yamashiro, Hirokazu Nishiwaki, Shinichiro J. Tojo, Yuichiro Ogura, Yoshihito Honda; In Vivo Evaluation of Platelet–Endothelial Interactions in Retinal Microcirculation of Rats. Invest. Ophthalmol. Vis. Sci. 1999;40(12):2918-2924.

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Abstract

purpose. This study was designed to develop a new method to evaluate the dynamics of platelets in the retinal microcirculation in vivo and to investigate quantitatively the platelet–endothelial interactions in rat retina with the use of this system.

methods. Isolated platelet samples were labeled with carboxyfluorescein diacetate succinimidyl ester. After intravenous administration, platelet behavior in the retinal microcirculation was evaluated with a scanning laser ophthalmoscope. The images were recorded on S-VHS videotape and analyzed with a computer-assisted image analysis system. The platelet–endothelial interactions in the retinal microcirculation were also investigated with the use of lipopolysaccharide-stimulated endothelium or platelets activated with thrombin.

results. Fluorescent platelets were recognized as distinct dots in the retinal microcirculation and could be traced frame by frame. The velocity of platelets in the retinal arteries, capillaries, and veins was 26.1 ± 6.4, 1.6 ± 0.4, and 19.9 ± 8.2 mm/sec, respectively. In control rats, even the activated platelets showed minimal interaction with retinal endothelial cells. In contrast, stimulated retinal endothelium showed active platelet–endothelial interactions; many platelets were observed rolling and adhering along the major retinal veins. The interactions between platelets and stimulated endothelial cells were substantially inhibited with the injection of P-selectin monoclonal antibody.

conclusions. The present study demonstrated a new method to visualize platelet behavior in the retinal microcirculation in vivo. This method will allow quantitative evaluation of platelet dynamics and platelet–endothelial interactions in retinal pathologic conditions.

Platelets undoubtedly play a leading role in hemostasis and thrombus formation. 1 Previous experimental studies have shown that the process of thrombus formation in the flowing blood is mediated differently from that in static situations. 2 3 Recently, Savage et al. 4 have indicated that two adhesion molecules, glycoprotein(GP)IIb-IIIa and GPIb, and two distinct substrates, fibrinogen and von Willebrand factor, are required to stabilize platelet adhesion under various types of shear stress. Whereas GPIIb-IIIa is essential for firm adhesion of platelets, GPIb is first required to tether the platelets contacting the surface under high-flow conditions, thereby reducing their velocity and facilitating their firm adhesion through GPIa-IIa. 
In addition, it is suggested that platelets are involved in the pathogenesis of many inflammatory conditions 5 such as ischemia–reperfusion injury. 6 On activation, accumulated platelets produce free radicals and proinflammatory mediators such as serotonin, leukotrienes, thromboxane A2, and platelet-derived growth factor. 7 Moreover, platelets have a potential to modulate leukocyte functional response. 8 To recruit flowing platelets to the inflammatory region, it is necessary for platelets to interact with the endothelial cells through distinct adhesion molecules expressed on the surface of platelets and endothelial cells. 9 10 Recently, an intravital microscopic study reported that platelets can roll on postischemic endothelium through P-selectin, in the course of accumulation during ischemia–reperfusion injury. 11  
During the past decade, large advances have been made in the research of interactions between leukocytes and retinal endothelial cells. 12 13 14 15 This research is supported by biochemical and histologic investigations about adhesion molecules on the surface of leukocytes and endothelial cells. 16 17 In spite of the progress in experiments in vitro of platelet–endothelial interactions, 7 we believe that no information is available about interactions between platelets and retinal endothelium in vivo. 18 19 In this study, we developed a new method to visualize and evaluate the dynamics of platelets in the retinal microcirculation. With the use of this system, we investigated platelet–endothelial interactions in rat retina. 
Materials and Methods
Animal
Male pigmented Long–Evans rats and female albino Lewis rats (200–250 g) were used to observe the fundus and provide the blood samples in this study. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Platelet Preparation
Carboxyfluorescein diacetate succinimidyl ester (CFDASE; Molecular Probes, Eugene, OR) is a nonfluorescent precursor that diffuses into cells and forms a stable fluorochrome carboxyfluorescein succinimidyl ester (CFSE; peak absorbance, 492 nm; peak emission, 518 nm) after catalysis by esterase. This enzymatic reaction occurs predominantly in leukocytes and platelets and partially in serum. Intracellular fluorophores react with lysine residues of protein and remain within the cell as long as the membrane is intact. 20  
CFDASE was dissolved in dimethyl sulfoxide (Wako Pure Chemicals, Osaka, Japan) to a concentration of 15.6 mM, and a small aliquot (200 μl) was stored at −70°C until use. 
Blood samples from donor rats of either strain were harvested from the abdominal artery and collected in polypropylene tubes containing 2 ml volume of 38 mOsM citric acid, 75 mM trisodium citrate, 100 mOsM dextrose. The blood was centrifuged at 250g for 10 minutes. 11 Platelet–rich plasma was gently transferred to a fresh tube and centrifuged at 2000g for 10 minutes. The platelet pellet was resuspended in 20 ml Hanks’ balanced salt solution (Gibco, Grand Island, NY) and incubated with 100 μl CFDASE solution for 30 minutes at 37°C. 21 After incubation, the platelet suspension was centrifuged again at 2000g for 10 minutes. 11 The platelet pellet was resuspended in Hanks’ balanced salt solution at a concentration of 1 × 106 platelets/0.3 ml or 2 × 108 platelets/0.3 ml. 
Experimental Procedure
Each rat of either strain was anesthetized with xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (10 mg/kg). The pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride. A contact lens was used to retain corneal clarity throughout the experiment. Each rat had a catheter inserted into the tail vein and was placed on a stereotaxic platform. Arterial blood pressure and heart rate were monitored with the blood pressure analyzer (IITC, Woodland Hills, CA). Platelets (1 × 106) were infused to measure the velocity of each platelet in the retinal microcirculation, and 2 × 108 platelets were used to evaluate the interactions with the retinal endothelial cells. A low dose of CFSE solution (Molecular Probes) was administered to delineate the retinal vasculature. The fundus was observed with a scanning laser ophthalmoscope (SLO; Rodenstock, Munich, Germany) in the 20° or 40° field. The argon blue laser (wavelength, 488 nm) was used for the illumination source, with a regular emission filter for fluorescein angiography. The obtained images were recorded on an S-VHS videotape at the video rate of 30 frames/sec for further analysis. 
Experimental Design
The endothelial cells of retinal vessels in recipient rats or platelet samples from donor rats were activated in some experiments to investigate the platelet–endothelial interactions in the retinal microcirculation. To stimulate the endothelial cells of the retinal vessels, 200 μg lipopolysaccharide (LPS) from Salmonella typhimurium (Difco, Detroit, MI) was injected into the footpad of the recipient rats. In this study, recipient rats were used at 12 hours after LPS treatment. 12 To activate platelets, purified platelets were incubated with 0.2 U/ml thrombin (Sigma, St. Louis, MO) for 15 minutes at 37°C after labeling with CFDASE. 9 10  
As a control, six female Lewis rats were administered nonactivated platelets (group 1). To investigate the effects of activation of endothelial cells on the platelet–endothelial interactions, nonactivated platelets were injected into six female Lewis rats treated with LPS (group 3). Activated platelets were injected into the nonsurgical Lewis rats (group 2; n = 6) or Lewis rats treated with LPS (group 4; n = 6). To evaluate the involvement of P-selectin in platelet–endothelial interactions, 2 mg/kg anti-rat P-selectin monoclonal antibody (mAb) ARP2-4 22 was administered intravenously to the female Lewis rats treated with LPS, 5 minutes before injection of nonactivated platelets (group 5; n = 6). 
Image Analysis
The video recordings were analyzed with an image analysis system, consisting of a personal computer (Apple Computer, Cupertino, CA) equipped with a video digitizer (Radius, San Jose, CA). The latter digitizes the video image in real time (30 frame/sec) to 640 horizontal and 480 vertical pixels with an intensity resolution of 256 steps. We investigated the behavior of platelets in the retinal vessels to evaluate the platelet–endothelial interactions. Rolling platelets were defined as platelets that moved at a slower velocity than free-flowing platelets in a given vessel and that made intermittent adhesive contacts with vascular endothelial cells. 9 10 11 12 13 14 15 A platelet was defined as adherent to vascular endothelium if it remained stationary for longer than 30 seconds. 11 The number of rolling platelets was calculated as the total number of rolling platelets along all major veins for 1 minute within a circle with a radius of 500 μm from the center of the optic disc. Velocity of rolling platelets was calculated as the time required for a platelet to travel a given distance along the vessel. 9 10 11 12 13 14 15 The number of platelets adhering to the venous endothelial walls was quantified within the same circle. All parameters were evaluated after a stabilization period of 5 minutes after the administration of platelets. 
After these experiments, the rats were killed with an overdose of anesthesia and the eyes enucleated to determine a calibration factor with which to convert values measured on a computer monitor (in pixels) into real values (in micrometers). 
Statistical Analysis
All values are presented as means ± SD. The data were analyzed using a one-way analysis of variance with Scheffé’s post hoc test. Differences were considered statistically significant at P < 005. 
Results
Platelet Behavior in the Retinal Microcirculation
Immediately after platelet suspension was infused intravenously, flowing platelets were recognized as distinct dots in the retinal microcirculation. When 2 × 108 platelets were infused, it was difficult to trace the track of each platelet, especially in the major retinal arteries and veins (Fig. 1) . In rats administered 1 × 106 platelets, however, the movement of each platelet could be followed frame by frame. With analysis of every frame, the velocity of platelets could be calculated from the artery through the capillary to the vein (Fig. 2) . The velocity of platelets in the retinal arteries, capillaries, and veins were 26.1 ± 6.4, 1.6 ± 0.4, and 19.9 ± 8.2 mm/sec, respectively. No adverse effects on heart rate or blood pressure were observed on infusion of fluorescent platelets. 
In the control rats, all the fluorescent platelets were observed freely flowing in the major retinal arteries and veins. In LPS-treated rats, among the many free-flowing platelets, some platelets were observed slowly rolling along the major retinal veins (Fig. 3) . Figure 4 indicates the velocity of all fluorescent platelets in a major retinal vein of a control and an LPS-treated rat. Because the velocity of rolling platelets was at least an order of magnitude lower than those of free-flowing platelets in the major retinal veins, it was not difficult to distinguish rolling platelets from free-flowing platelets on a video monitor. Moreover, some platelets adhered to the vessel walls of the capillary and major retinal veins and even to the major retinal arteries. 
Platelet–Endothelial Interaction in the Retina
Figure 5 shows the numbers of rolling and adhering fluorescent platelets along the major retinal veins in each group. In a control rat infused with nonactivated platelets (group 1), although a small number of platelets were captured in the retinal capillaries, no platelets were observed rolling or adhering to the major retinal veins. Similarly, even if infused with activated platelets (group 2), control rats showed minimal changes in platelet–endothelial interactions in the retina. In contrast, stimulated retinal endothelium showed active platelet–endothelial interactions. Many platelets were observed rolling and adhering along the major retinal veins (group 3). The velocity of rolling platelets along the major retinal veins was 34.8 ± 17.3 μm/sec. However, no more intimate interactions were recognized, even if activated platelets were infused into the LPS-treated rats. In contrast, interactions between platelets and stimulated endothelial cells were substantially inhibited by treatment with P-selectin mAb ARP2-4. In group 5, the number of rolling and adhering platelets along the major retinal veins was reduced to 12.4% and 36.8%, with inhibition of P-selectin (P < 0.001 and P = 0.039, compared with values in group 3). 
Thrombosis Formation in the Retinal Arteries
In three arteries of two rats, in which retinal endothelial cells were stimulated with LPS, increasing numbers of platelets were observed adhering focally to the arterial endothelial walls in a line forming a thrombus (Fig. 6 A). Although, even in the LPS-stimulated rats, no rolling platelets were observed along the major retinal arteries, many platelets moving in a marginal flow were tethered for a moment to the thrombus on the arterial walls. After that, most platelets flowed away; however, some platelets firmly adhered, enlarging the thrombus on the arterial walls. Meanwhile, the remaining thrombi flowed away downstream except for one, which flowed into the capillary to obstruct the capillary blood flow (Fig. 6B)
Discussion
In this study, we developed a new method to visualize and evaluate quantitatively the platelet movement in the retinal microcirculation in vivo with the use of CFDASE. CFDASE is a nonfluorescent precursor that diffuses into cells and forms a stable fluorochrome, CFSE, after catalysis by esterase. 23 This dye has been widely used to observe lymphocyte migration 21 and to track neuronal cells 24 and engrafted hepatocytes. 25 Accumulating reports have indicated that CFDASE does not affect the viability of labeled cells. 20 21 24 25 26 Also, CFDASE is so lipophilic that it easily permeates the membrane and, after catalysis, reacts with lysine residues of proteins and remains within the cell as long as the cell is alive. 20 Therefore, it allows us to observe for a long period, as Karrer et al. 26 demonstrated with transplanted hepatocytes labeled with CFDASE in recipient rats for up to 20 days. Moreover, although many fluorescent dyes bind to the cell membrane, 18 19 which may interfere with the epitopes of the cell surfaces, CFDASE, which binds to intracellular molecules, would have minimal effect on in vivo platelet–endothelial interactions. 25 In addition, we administered 2 × 108 fluorescent platelets to each rat. The number of peripheral blood platelets of the control rats was 9.9 ± 2.1 × 105/μl. Assuming that the blood volume of rats is 6 ml/100g body weight, 10 we estimate that infused platelets were not more than 2% of the circulating platelets. Taken together, undesirable effects on the microcirculation would be limited in administering fluorescent platelets. 
To our knowledge, little information is currently available about the velocity of platelets in retinal microcirculation. 18 In this study, the velocity of platelets in the retinal arteries, veins, and capillaries in control rats was 26.1 ± 6.4, 19.9 ± 8.2, and 1.6 ± 0.4 mm/sec, respectively. In a previous study with acridine orange digital fluorography, Nishiwaki et al. 27 reported that the velocity of leukocytes was 29.5 ± 7.3, 17.4 ± 5.3, and 1.4 ± 0.4 mm/sec in the major retinal arteries, veins, and capillaries, respectively. Their results were relatively comparable with our current findings. However, as they discussed, digital processing used in the SLO is restricted spatially and temporally. 27 In major retinal vessels, especially in arteries, fluorescent particles sometimes disappear when constitutive frames are analyzed. It is possible that the movement of the fastest platelets could not be traced consecutively, because the video rate was 30 frames/sec. Otherwise, because the SLO uses an interlaced scanning system, 28 the SLO may not have been able to detect the figure of a platelet between two lines of the scanning laser. 29 Moreover, the velocity of platelets depends on the pulsatile nature of the flow in the artery or on the cell location across the vessels in the artery or vein. 30 Blood cells flowing closer to the axis flow faster compared with those in the marginal stream. 31 Therefore, there is a limitation in measuring the velocity of platelets in the major retinal vessels. In contrast, the velocity of platelets in the capillaries is comparable with that of leukocytes. Leukocytes, which are larger than the caliber of the capillary, have to be squeezed during passage through the narrow capillary, accompanied by platelets and erythrocytes in rouleaux formations. 32 This accounts for the similarity of velocity in the capillaries between platelets and leukocytes. 
It has been suggested that P-selectin mediates platelet–endothelial interactions, 9 10 11 and Zachem et al. 5 have recently shown that P-selectin mAb attenuates platelet accumulation in glomerulonephritis. In this study, we investigated platelet–endothelial interactions in the retinal microcirculation with the use of LPS-stimulated endothelium or platelets activated with thrombin. Thrombin can activate platelets and induce rapid expression of P-selectin on their surface within a few minutes. 33 Under basal conditions of the endothelium, even activated platelets showed minimal interactions with retinal endothelial cells. These findings indicate that activation of platelets had a minor influence on platelet–endothelial interactions. Perhaps, nitric oxide or prostaglandin I2 derived from endothelial cells contributes partially to the antiplatelet property of the endothelium. 34 In contrast, retinal endothelial cells stimulated with LPS showed active interactions with platelets, similar to the findings of Miyamoto et al., 12 who showed leukocytes rolling along retinal veins in LPS-treated rats, with a peak 12 hours after induction. Although injected platelets may also be activated with various kinds of cytokines induced by LPS treatment immediately after intravenous infusion, 7 certainly, retinal endothelial cells were activated with the treatment of LPS and expressed P-selectin on their surfaces. 15 P-selectin expression on the endothelial cells is essential for platelet rolling on the venous walls. 10 11 Our current observation has been supported by an intravital microscopic study of Frenette et al. 10 of P-selectin–deficient mice. These investigators have shown that platelets from P-selectin–deficient and wild-type mice can roll along the stimulated endothelium of wild-type mice but not along the stimulated endothelium of P-selectin–deficient mice. P-selectin, which is stored in Weibel–Palade bodies of endothelial cells, modulate the recruitment of not only leukocytes but also platelets to the inflammatory regions. 35  
In another study of P-selectin–deficient mice, Massberg et al. 11 observed platelet rolling on the arterial and venous walls in the postischemic mesentery. In our study, no platelet rolling was observed along the major retinal arteries. However, we demonstrated thrombus formation and platelet tethering in the retinal arteries. The expression of P-selectin is reported to be limited substantially on the arterial endothelium, compared with that on the veins. 35 Moreover, in the major retinal artery 10 to 50 μm in diameter, platelet adhesion is rarely observed because of the high shear stress. 4 30 Therefore, this interaction is mediated by other adhesion molecules. 2 3 As Savage et al. 4 have recently suggested, various kinds of adhesion molecules work together in the thrombus formation under high-flow conditions in the retinal arteries. 
In conclusion, we developed a new method to evaluate quantitatively the platelet dynamics and platelet–endothelial interactions in the retinal microcirculation in vivo. In platelet adhesion to vascular walls, it is necessary for flowing platelets to interact with the endothelial cells through distinct adhesion molecules expressed on the surface of platelets and endothelial cells. 9 10 11 In this study, activation of platelets had a minor influence on platelet–endothelial interactions; however, endothelial cells treated with LPS showed active interactions with platelets. P-selectin expressed on the endothelial cells modulated the recruitment of platelets to the inflamed retina. 
 
Figure 1.
 
A fluorescent fundus image of a control rat after administration of 2 × 108 fluorescently labeled platelets. Fluorescent platelets are visible in the artery, vein, and capillaries. The retinal vasculature is delineated with a low dose of CFSE solution.
Figure 1.
 
A fluorescent fundus image of a control rat after administration of 2 × 108 fluorescently labeled platelets. Fluorescent platelets are visible in the artery, vein, and capillaries. The retinal vasculature is delineated with a low dose of CFSE solution.
Figure 2.
 
(A) A digitized monochromatic fundus image obtained with SLO in a control rat. (B) A fluorescent fundus image of the same rat after administration of 1 × 106 fluorescently labeled platelets. The movement of each platelet could be followed frame by frame (arrowhead). (C) A composite image of movement of a single fluorescent platelet shown in (B) as it traveled from the artery through the capillary to the vein. (D) The velocity was calculated by analyzing the movement between consecutive fluorescent dots shown in (C).
Figure 2.
 
(A) A digitized monochromatic fundus image obtained with SLO in a control rat. (B) A fluorescent fundus image of the same rat after administration of 1 × 106 fluorescently labeled platelets. The movement of each platelet could be followed frame by frame (arrowhead). (C) A composite image of movement of a single fluorescent platelet shown in (B) as it traveled from the artery through the capillary to the vein. (D) The velocity was calculated by analyzing the movement between consecutive fluorescent dots shown in (C).
Figure 3.
 
A digitized monochromatic fundus image of an LPS-treated rat (top). A sequence of fluorescent fundus images of the same rat after administration of fluorescent platelets (bottom). The retinal vasculature is delineated slightly with a low dose of CFSE solution. Arrows indicate a platelet rolling along a major retinal vein; arrowheads indicate two free-flowing platelets. The second picture from the top represents the image at 1 frame (0.033 seconds) after the first picture. The third and fourth pictures represent the images at 30 and 300 frames (1 and 10 seconds) after the second picture. It was not difficult to discriminate rolling platelets from free-flowing platelets on a video monitor, because their velocities were markedly different.
Figure 3.
 
A digitized monochromatic fundus image of an LPS-treated rat (top). A sequence of fluorescent fundus images of the same rat after administration of fluorescent platelets (bottom). The retinal vasculature is delineated slightly with a low dose of CFSE solution. Arrows indicate a platelet rolling along a major retinal vein; arrowheads indicate two free-flowing platelets. The second picture from the top represents the image at 1 frame (0.033 seconds) after the first picture. The third and fourth pictures represent the images at 30 and 300 frames (1 and 10 seconds) after the second picture. It was not difficult to discriminate rolling platelets from free-flowing platelets on a video monitor, because their velocities were markedly different.
Figure 4.
 
The distribution of the velocity of consecutive fluorescent platelets flowing in a given vein in a control rat (A) and in an LPS-treated rat (B). All fluorescent platelets were free-flowing in the control rat. In the LPS-treated rats, among many free-flowing platelets, some leukocytes were observed moving extremely slowly compared with other fluorescent platelets. (C) The distribution of the velocity of extremely slow-moving (rolling) platelets is shown in (B). The velocity of rolling platelets was at least an order of magnitude lower than those of other free-flowing platelets.
Figure 4.
 
The distribution of the velocity of consecutive fluorescent platelets flowing in a given vein in a control rat (A) and in an LPS-treated rat (B). All fluorescent platelets were free-flowing in the control rat. In the LPS-treated rats, among many free-flowing platelets, some leukocytes were observed moving extremely slowly compared with other fluorescent platelets. (C) The distribution of the velocity of extremely slow-moving (rolling) platelets is shown in (B). The velocity of rolling platelets was at least an order of magnitude lower than those of other free-flowing platelets.
Figure 5.
 
The numbers of rolling (A) and adherent (B) platelets along the major retinal veins in each group. Values are means ± SD. *P < 0.01 compared with values of control rats injected with nonactivated platelets (group 1); P < 0.01, P< 0.05, compared with values of LPS-treated rats injected with non-activated platelets (group 3).
Figure 5.
 
The numbers of rolling (A) and adherent (B) platelets along the major retinal veins in each group. Values are means ± SD. *P < 0.01 compared with values of control rats injected with nonactivated platelets (group 1); P < 0.01, P< 0.05, compared with values of LPS-treated rats injected with non-activated platelets (group 3).
Figure 6.
 
(A) A fluorescent fundus image of an LPS-treated rat administered fluorescently labeled platelets shows a thrombus on the arterial wall. Although many platelets flowing in a marginal flow were tethered for a moment to the thrombus, this was difficult to show in a still picture. (B) A sequence of four successive (frame-by-frame) fundus images of a different LPS-treated rat administered fluorescently labeled platelets. A thrombus formed on an arterial wall, but it suddenly flowed away downstream.
Figure 6.
 
(A) A fluorescent fundus image of an LPS-treated rat administered fluorescently labeled platelets shows a thrombus on the arterial wall. Although many platelets flowing in a marginal flow were tethered for a moment to the thrombus, this was difficult to show in a still picture. (B) A sequence of four successive (frame-by-frame) fundus images of a different LPS-treated rat administered fluorescently labeled platelets. A thrombus formed on an arterial wall, but it suddenly flowed away downstream.
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Figure 1.
 
A fluorescent fundus image of a control rat after administration of 2 × 108 fluorescently labeled platelets. Fluorescent platelets are visible in the artery, vein, and capillaries. The retinal vasculature is delineated with a low dose of CFSE solution.
Figure 1.
 
A fluorescent fundus image of a control rat after administration of 2 × 108 fluorescently labeled platelets. Fluorescent platelets are visible in the artery, vein, and capillaries. The retinal vasculature is delineated with a low dose of CFSE solution.
Figure 2.
 
(A) A digitized monochromatic fundus image obtained with SLO in a control rat. (B) A fluorescent fundus image of the same rat after administration of 1 × 106 fluorescently labeled platelets. The movement of each platelet could be followed frame by frame (arrowhead). (C) A composite image of movement of a single fluorescent platelet shown in (B) as it traveled from the artery through the capillary to the vein. (D) The velocity was calculated by analyzing the movement between consecutive fluorescent dots shown in (C).
Figure 2.
 
(A) A digitized monochromatic fundus image obtained with SLO in a control rat. (B) A fluorescent fundus image of the same rat after administration of 1 × 106 fluorescently labeled platelets. The movement of each platelet could be followed frame by frame (arrowhead). (C) A composite image of movement of a single fluorescent platelet shown in (B) as it traveled from the artery through the capillary to the vein. (D) The velocity was calculated by analyzing the movement between consecutive fluorescent dots shown in (C).
Figure 3.
 
A digitized monochromatic fundus image of an LPS-treated rat (top). A sequence of fluorescent fundus images of the same rat after administration of fluorescent platelets (bottom). The retinal vasculature is delineated slightly with a low dose of CFSE solution. Arrows indicate a platelet rolling along a major retinal vein; arrowheads indicate two free-flowing platelets. The second picture from the top represents the image at 1 frame (0.033 seconds) after the first picture. The third and fourth pictures represent the images at 30 and 300 frames (1 and 10 seconds) after the second picture. It was not difficult to discriminate rolling platelets from free-flowing platelets on a video monitor, because their velocities were markedly different.
Figure 3.
 
A digitized monochromatic fundus image of an LPS-treated rat (top). A sequence of fluorescent fundus images of the same rat after administration of fluorescent platelets (bottom). The retinal vasculature is delineated slightly with a low dose of CFSE solution. Arrows indicate a platelet rolling along a major retinal vein; arrowheads indicate two free-flowing platelets. The second picture from the top represents the image at 1 frame (0.033 seconds) after the first picture. The third and fourth pictures represent the images at 30 and 300 frames (1 and 10 seconds) after the second picture. It was not difficult to discriminate rolling platelets from free-flowing platelets on a video monitor, because their velocities were markedly different.
Figure 4.
 
The distribution of the velocity of consecutive fluorescent platelets flowing in a given vein in a control rat (A) and in an LPS-treated rat (B). All fluorescent platelets were free-flowing in the control rat. In the LPS-treated rats, among many free-flowing platelets, some leukocytes were observed moving extremely slowly compared with other fluorescent platelets. (C) The distribution of the velocity of extremely slow-moving (rolling) platelets is shown in (B). The velocity of rolling platelets was at least an order of magnitude lower than those of other free-flowing platelets.
Figure 4.
 
The distribution of the velocity of consecutive fluorescent platelets flowing in a given vein in a control rat (A) and in an LPS-treated rat (B). All fluorescent platelets were free-flowing in the control rat. In the LPS-treated rats, among many free-flowing platelets, some leukocytes were observed moving extremely slowly compared with other fluorescent platelets. (C) The distribution of the velocity of extremely slow-moving (rolling) platelets is shown in (B). The velocity of rolling platelets was at least an order of magnitude lower than those of other free-flowing platelets.
Figure 5.
 
The numbers of rolling (A) and adherent (B) platelets along the major retinal veins in each group. Values are means ± SD. *P < 0.01 compared with values of control rats injected with nonactivated platelets (group 1); P < 0.01, P< 0.05, compared with values of LPS-treated rats injected with non-activated platelets (group 3).
Figure 5.
 
The numbers of rolling (A) and adherent (B) platelets along the major retinal veins in each group. Values are means ± SD. *P < 0.01 compared with values of control rats injected with nonactivated platelets (group 1); P < 0.01, P< 0.05, compared with values of LPS-treated rats injected with non-activated platelets (group 3).
Figure 6.
 
(A) A fluorescent fundus image of an LPS-treated rat administered fluorescently labeled platelets shows a thrombus on the arterial wall. Although many platelets flowing in a marginal flow were tethered for a moment to the thrombus, this was difficult to show in a still picture. (B) A sequence of four successive (frame-by-frame) fundus images of a different LPS-treated rat administered fluorescently labeled platelets. A thrombus formed on an arterial wall, but it suddenly flowed away downstream.
Figure 6.
 
(A) A fluorescent fundus image of an LPS-treated rat administered fluorescently labeled platelets shows a thrombus on the arterial wall. Although many platelets flowing in a marginal flow were tethered for a moment to the thrombus, this was difficult to show in a still picture. (B) A sequence of four successive (frame-by-frame) fundus images of a different LPS-treated rat administered fluorescently labeled platelets. A thrombus formed on an arterial wall, but it suddenly flowed away downstream.
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