March 2004
Volume 45, Issue 3
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Retina  |   March 2004
Platelets Adhering to the Vascular Wall Mediate Postischemic Leukocyte–Endothelial Cell Interactions in Retinal Microcirculation
Author Affiliations
  • Kazuaki Nishijima
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Junichi Kiryu
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Akitaka Tsujikawa
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Kazuaki Miyamoto
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Megumi Honjo
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Hidenobu Tanihara
    Department of Ophthalmology, Kumamoto University, Kumamoto, Japan; and the
  • Atsushi Nonaka
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Kenji Yamashiro
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Hideto Katsuta
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Shinsuke Miyahara
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Yoshihito Honda
    From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; the
  • Yuichiro Ogura
    Department of Ophthalmology, Nagoya City University Medical School, Nagoya, Japan.
Investigative Ophthalmology & Visual Science March 2004, Vol.45, 977-984. doi:10.1167/iovs.03-0526
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      Kazuaki Nishijima, Junichi Kiryu, Akitaka Tsujikawa, Kazuaki Miyamoto, Megumi Honjo, Hidenobu Tanihara, Atsushi Nonaka, Kenji Yamashiro, Hideto Katsuta, Shinsuke Miyahara, Yoshihito Honda, Yuichiro Ogura; Platelets Adhering to the Vascular Wall Mediate Postischemic Leukocyte–Endothelial Cell Interactions in Retinal Microcirculation. Invest. Ophthalmol. Vis. Sci. 2004;45(3):977-984. doi: 10.1167/iovs.03-0526.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Recent evidence suggests that platelets play a major role in ischemia–reperfusion injury, not only through thrombus formation but also through participation in inflammatory reactions with leukocytes. This study was designed to investigate the contribution of platelets in leukocyte recruitment to inflamed regions in vivo.

methods. Thrombocytopenia was produced in male Long-Evans rats by intravenous injection of anti-platelet serum at 4 hours before ischemia-reperfusion. Leukocyte behavior in retinal microcirculation was evaluated with acridine orange digital fluorography. Expression of P-selectin in the postischemia retina was investigated by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. After 14 days of reperfusion, ischemia-induced retinal damage was evaluated histologically.

results. Leukocyte rolling along major retinal veins of thrombocytopenic rats was dramatically suppressed, and subsequent leukocyte accumulation in the postischemia retina was also significantly reduced (72.3%; P < 0.001) at 24 hours after reperfusion. Although RT-PCR revealed no significant reduction of P-selectin mRNA in platelet-depleted rat retina after transient ischemia, immunohistologic examination showed suppression of P-selectin expression on the vascular wall. Another immunologic examination using anti-platelet antibody detected adherent platelets, which can also express P-selectin on their surfaces, on postischemic vascular endothelium in vehicle-treated retina. Moreover, blockage of platelet glycoprotein IIb/IIIa resulted in substantial inhibition of leukocyte rolling. In addition, histologic examination showed the participation of platelets in retinal ischemia–reperfusion injury.

conclusions. This study demonstrated that the expression of P-selectin on platelets may contribute to the recruitment of leukocytes to tissues after ischemia.

Inappropriate leukocyte infiltration into tissues after ischemia-reperfusion has been shown to exacerbate organ dysfunction. 1 2 3 This recruitment of leukocytes from the main stream of blood consists of a multistep cascade. 4 5 6 Emigration from the vasculature requires leukocyte tethering, rolling, and firm adhesion to the activated endothelial cells. In several investigations, including our previous studies in rat retina, 7 each step of leukocyte–endothelial cell interactions was mediated by a distinct adhesion molecule. The initial tethering and rolling events are mediated primarily by endothelial cell P-selectin, but activated β2-integrins are necessary for the subsequent firm attachment. 8 9 10 11 To date, platelets have been suggested to contribute to leukocyte recruitment to the vascular wall. Also, some investigators have demonstrated that activated platelets adherent to an intact endothelial cell monolayer in vitro bind flowing leukocytes, enabling them to transfer to the surface of the endothelium. 12 13  
Platelets may also play an important role in the pathogenesis of ischemia–reperfusion injury. 14 15 Platelets are known to be associated with tissue injury after ischemia-reperfusion through the formation of microthrombi 16 and by the release of various vasoactive substances. 17 18 19 During the past decade, many investigators have demonstrated that platelets are involved not only in the pathogenesis of thrombosis but also in that of inflammation. 20 21 22 Under normal physiologic conditions, platelets circulate without forming interactions with nonactivated vascular endothelium. However, once endothelial cells are activated, they exhibit active interactions with platelets flowing in the marginal blood flow. Massberg et al. 23 have shown that both nonactivated and activated platelets can roll in vivo on P-selectin expressed at the surface of activated endothelial cells in small mesenteric venules. In addition to P-selectin, glycoprotein IIb/IIIa (GP IIb/IIIa) is reported to be involved in platelet adhesion to endothelial cells through an Arg-Gly-Asp (RGD) peptide-dependent mechanism. 24 25 Adherent platelets on vascular endothelium release free radicals and inflammatory mediators, such as serotonin, leukotrienes, thromboxane A2, and platelet-derived growth factor. 18 19 Moreover, platelets are thought possibly to contribute to tissue injury indirectly by recruiting leukocytes to inflammatory regions and by modulating leukocyte functional responses in vivo. 26 27 28 However, little is known about the mechanism of platelet participation in leukocyte–endothelial cell interactions. 
In the study described herein, we investigated leukocyte dynamics in vivo by using the technique of acridine orange (AO) digital fluorography, a technique that allows us to visualize leukocyte behavior in the retinal microcirculation with minimal invasion. Using this technique in previous studies, we have found that leukocyte dynamics, such as rolling, adhesion, and accumulation, could be evaluated quantitatively in inflammatory conditions. 29 In addition, we have developed an in vivo method to evaluate platelet–endothelial cell interactions quantitatively in rat retina. 30 31 With the use of this method, we demonstrated earlier that platelets roll and adhere along the major retinal veins characterized by a high shear rate after transient retinal ischemia, and, furthermore, have shown that these interactions are mediated by endothelial cell P-selectin—not by platelet P-selectin. 31 In the present study, we used these two techniques to evaluate the role of platelets in leukocyte–endothelial cell interactions after retinal ischemia-reperfusion. 
Materials and Methods
Animal Model
Male pigmented Long-Evans rats (200–250 g) were used in the study. Rats were anesthetized with a mixture of xylazine hydrochloride and ketamine hydrochloride and the pupils dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride. Transient retinal ischemia for 60 minutes was induced in the right eye by ligation of the optic sheath under the operating microscope. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
AO Digital Fluorography
AO fluorography has been described previously. 32 Leukocytes were labeled with fluorescent nuclear dye of acridine orange (Wako Pure Chemical, Osaka, Japan) administered intravenously and then imaged with a scanning laser ophthalmoscope (SLO; Rodenstock Instruments, Munich, Germany). Arterial blood pressure and heart rate were monitored with a blood pressure analyzer (IITC, Woodland Hills, CA). The fundus was observed with the SLO in the 40° field for 5 minutes. At 30 minutes after injection of AO, the fundus was observed again to evaluate leukocytes that had accumulated in the retinal microcirculation. The images obtained were recorded for further analysis on an S-VHS videotape at a video rate of 30 frames/s. 
Evaluation of Leukocyte–Endothelial Interactions under Platelet-Depleted Conditions
Platelet-depleted rats were injected intravenously with rabbit anti-rat platelet serum (Inter-Cell Technologies, Hopewell, NJ) at 4 hours before ischemia induction. Vehicle-treated rats were given the same volume of rabbit anti-rat IgG. AO digital fluorography was performed in both platelet-depleted and nondepleted rats at 4, 12, and 24 hours after reperfusion. Nonischemia rats served as controls for each group. Six different animals were used at each time point. 
Evaluation of Platelet-Endothelial Cell Interactions
Platelet samples were prepared in accordance with the method described previously. 31 In brief, after blood from donor rats was centrifuged, platelets were extracted as a platelet pellet. The pellet was incubated with carboxyfluorescein diacetate succinimidyl ester. The platelet pellet was suspended in Hanks’ balanced salt solution (HBSS) at a concentration of 6 × 108 platelets/0.2 mL. 
Platelet behavior in the retinal microcirculation was evaluated at 4, 12, and 24 hours after reperfusion. Nonischemia rats were used as the control. Immediately before platelet administration, rats were anesthetized as described. Six different rats were used at each time point. A total of 6 × 108 platelets were administered, to evaluate their interactions with retinal endothelial cells. The fundus was observed by SLO with the argon blue laser and an emission filter typically used for fluorescein angiography. 
P-selectin and GP IIb/IIIa Involvement in Blood Cell–Endothelial Cell Interactions
The possible role of P-selectin and GP IIb/IIIa in blood cell–endothelial interactions was determined using anti-rat P-selectin mAb ARP2-4 (Sumitomo, Osaka, Japan) and Arg-Gly-Asp (RGD) peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP; Biomol, Hamburg, Germany). We divided the ischemic rats into three groups: vehicle-treated, ARP2-4-treated, and GRGDSP-treated. We administered 2 mg/kg of ARP2-4 or 12 mg/kg of purified GRGDSP peptide intravenously 5 minutes before reperfusion. At 12 hours after reperfusion, platelet and leukocyte behavior were evaluated. 
Image Analysis
The number of rolling leukocytes was calculated as the total number of rolling leukocytes per minute that crossed a fixed area of the veins at a distance of 1 disc diameter from the optic disc center. For evaluation of accumulated leukocytes, the number of fluorescent dots in the retina within 8 to 10 areas of 100 square pixels at a distance of 1 disc diameter from the edge of the optic disc were counted. The average number of dots of each individual area was used as the number of leukocytes accumulated in the retinal microcirculation for each rat. 
Rolling platelets were defined as platelets that moved at a slower velocity than that of free-flowing platelets in a given vessel and that made intermittent adhesive contacts with vascular endothelial cells. The number of rolling platelets was calculated as the total number of platelets rolling along each vein for 1 minute at a distance 1 disc diameter from the optic disc center. A platelet was defined as adherent to the vascular endothelium if it remained stationary for longer than 10 seconds. These were calculated as the total number of adherent platelets along all major retinal veins for 1 minute within a circle with a radius of 500 μm from the center of the optic disc. All parameters were evaluated after a stabilization period of 5 minutes after the administration of platelets. 
Semiquantification of P-selectin and ICAM-1 Gene Expression by Reverse Transcription–Polymerase Chain Reaction
To evaluate the effect of platelet depletion on expression of P-selectin and intercellular adhesion molecule (ICAM)-1 mRNA, eyes were enucleated at 6, 12, and 24 hours after reperfusion. Six rats were used at each time point. Each enucleated eye was cut into two pieces along the limbus, and the retina was then collected from the posterior segment. Nonischemia eyes were used as the control. Total RNA was isolated from the retina according to the acid guanidinium thiocyanate-phenol-chloroform extraction method. The extracted RNA was quantified, and then 5 μg of the RNA was subjected to RT-PCR analysis as described. 33  
Immunohistochemical Procedures
At 12 hours after reperfusion, rats were perfusion fixed with 4% paraformaldehyde before eyes were enucleated. Subsequently, the enucleated eyes were further fixed for 2 hours at 4°C in 4% paraformaldehyde and washed in phosphate-buffered saline (PBS). Each enucleated eye was cut into two pieces along the limbus and then gently shaken overnight at 4°C in 15% sucrose/PBS. The eye cups were embedded in optimal cutting temperature (OCT; Tissue-Tek; Miles, Inc., Elkhart, IN) and frozen on powdered dry ice. Sections (10 μm) were cut on a cryostat and collected onto silanized slides (Dako Japan, Kyoto, Japan). 
Retinal sections were washed twice for 3 minutes each in PBS and then incubated in 5% skim milk with 10% normal goat serum in PBS for 30 minutes and a solution of antibodies to P-selectin (ARP2-4, 1:1000 dilution in blocking solution) overnight at 4°C. After washing in PBS, they were treated for 30 minutes with Cy5-conjugated secondary antibodies (Chemicon, Temecula, CA) diluted 1:100. For double staining with von Willebrand factor (vWF) as a marker for vascular endothelium, the sections were incubated with a solution of antibody to vWF (Dako Japan, 1:400 dilution in blocking solution) and Cy3-conjugated secondary antibody (Chemicon). Similarly, the sections were incubated with a solution of rabbit anti-rat thrombocyte antibody (1:200 dilution) and Cy3-conjugated secondary antibody to stain the platelets adhering to the postischemia vascular endothelium. Sections were mounted and then observed under a confocal microscope (LSM410; Carl Zeiss Meditec, Oberkochen, Germany). 
Histologic Procedures
Six eyes from six rats each in the platelet-depleted, vehicle-treated, and nonsurgical control groups were obtained to evaluate the severity of retinal damage. After 14 days of reperfusion, the rats were killed with an anesthetic overdose. The surgically treated eyes were immediately enucleated and fixed in 1.48% formaldehyde and 1% glutaraldehyde in phosphate buffer and then in 3.7% formaldehyde. The eyes were dehydrated, embedded in paraffin, sectioned with a microtome at 2-μm thickness, and stained with hematoxylin and eosin. Each section was cut along the horizontal meridian of the eye through the optic nerve head. Sections were cut perpendicular to the retinal surface. Retinal sections were examined with an optical microscope (×400) by a masking procedure and then digitized by a charge-coupled device camera on a computer monitor. 
To quantify the retinal damage induced by ischemia–reperfusion injury, we measured changes in thickness of the retina. 34 The thickness of the inner plexiform layer (IPL), inner nuclear layer (INL), outer nuclear layer (ONL), and the overall retina from outer to inner limiting membrane (ILM-OLM) was measured. The thickness of the various retinal layers in each section was measured at a distance of 1.5 mm from the center of the optic nerve head. Each retinal thickness was averaged from 10 measurements of four sections from each eye. 
Statistical Analysis
All values are presented as mean ± SEM. Student’s t-test was used to compare two groups. ANOVA was used to compare three or more conditions, with post hoc comparisons tested using the Fisher protected least-significant difference procedure. Differences were considered statistically significant at P < 0.05. 
Results
Platelet Depletion with Anti-Platelet Serum
Figure 1A indicates platelet counts in the peripheral blood by elapsed time after the administration of anti-platelet serum (APS). The number of peripheral platelets, which was reduced immediately after injection of APS, remained low (<90%, P < 0.0001) until 24 hours after injection. At 48 hours after administration, however, it was increased to 70% of control rat levels. 
Leukocyte–Endothelial Cell Interactions in Postischemia Retinal Microcirculation
Immediately after the intravenous administration of AO solution, leukocytes were stained selectively among circulating blood cells. Among many free-flowing leukocytes labeled with AO, some were rolling slowly along the major retinal veins in postischemia eyes (Fig. 2A) . In the major retinal arteries, however, no rolling leukocytes were observed throughout the experiments. In vehicle-treated rats, the number of rolling leukocytes increased substantially after reperfusion and reached a peak at 12 hours. In platelet-depleted rats, leukocyte rolling was significantly inhibited (Fig. 2B , P < 0.0001). The number of rolling leukocytes in platelet-depleted rats were significantly reduced by 97% (P = 0.0037) and 96% (P = 0.0019) at 12 and 24 hours, respectively, after reperfusion compared with those in vehicle-treated rats. In the control rats, no leukocyte rolling was seen along the major retinal veins. 
Figures 2C and 2D show the changes in the number of accumulated leukocytes in the postischemia retinal microcirculation. In nonsurgical control rats, only a few leukocytes were recognized in the retinal microcirculation, whereas in the surgical groups accumulated leukocytes began to increase on at 4 hours after reperfusion and subsequently increased at 24 hours. However, leukocyte accumulation in the postischemia retina was significantly inhibited with treatment by APS (P < 0.0001). The number of accumulated leukocytes in postischemia retinas was reduced by 43% (P = 0.025) and 72% (P < 0.0001) at 12 and 24 hours, respectively, after reperfusion. 
Platelet–Endothelial Cell Interactions in Postischemia Retina
Immediately after the labeled platelets were infused intravenously, they were visible as distinct fluorescent dots circulating in the retinal microcirculation. In the control rats, no platelets actively interacted with retinal endothelial cells. In the postischemia retina, however, some platelets, among many free-flowing platelets, were observed slowly rolling or tethered along major retinal veins. Most platelets rolling along the postischemia retinal veins were observed away from the optic disc or flowing downstream. Others showed decreased velocity and were adhering to the vascular walls. The heart rate and blood pressure did not change significantly after the injection of labeled platelets (Table 1) . The number of rolling and adherent platelets along major retinal veins increased substantially in the postischemia retinas, reached a peak at 12 hours after reperfusion, and decreased to almost basal levels at 48 hours (Figs. 3A 3B)
Intravenous administration of ARP2-4 and GRGDSP significantly attenuated platelet adhesion along the postischemia veins (Fig. 4) . The numbers of rolling platelets in the ARP2-4- and GRGDSP-treated rats were reduced by 40.2% (P = 0.025) and 76.5% (P = 0.0004), respectively, compared with the number in the vehicle-treated group. The number of adherent platelets along the veins of ARP2-4- and GRGDSP-treated rats was reduced by 77.2% (P = 0.0007) and 79.5% (P = 0.0009). In addition, leukocyte rolling in both treated groups was reduced by 83.0% (P = 0.0058) and 71.4% (P = 0.025). 
Gene Expression of Adhesion Molecules in the Retina
At 6, 12, and 24 hours after reperfusion, P-selectin mRNA expression was upregulated in postischemia retinas (20- to 32-fold), but there was no significant difference in the expression of P-selectin mRNA in platelet-depleted retinas after ischemia-reperfusion (P > 0.05). In addition, ICAM-1 mRNA expression in the surgically treated rats was 23 to 26 times greater than that in control rats, but was not suppressed in platelet-depleted rats. 
Immunostaining Studies
At 12 hours after reperfusion, immunostaining for vWF and P-selectin was performed in retinal specimens from control, vehicle-treated, and platelet-depleted postischemia rats. Immunostaining was absent on all sections incubated without the primary antibody. On sections incubated with vWF antibody, immunoreactivity was present predominantly in the venous endothelium of the retinas of all rats. Intense P-selectin immunoreactivity was present in the venous endothelium from vehicle-treated rats, whereas only faint immunoreactivity was seen in that from platelet-depleted rats (Fig. 5)
In addition, immunoreactivity was intense along the vascular endothelium after ischemia-reperfusion on the sections incubated with anti-platelet polyclonal antibody (Fig. 6) , whereas no immunoreactivity was seen in nonischemia control sections. In some sections, leukocytes were observed adhering to vascular endothelium. 
Retinal Damage
Histologic examination showed destruction of the retinal structures in surgical rats, resulting in a decrease in retinal thickness and damage of retinal cells (Fig. 7A) . Figure 7B shows the thickness of each retinal layer at 14 days after reperfusion. The decrease in retinal thickness was more severe in the inner retina than in the outer retina. Thicknesses of the IPL, INL, and ILM-OLM in vehicle-treated rats (30%, 62%, and 60% that of nonsurgical rats, respectively) were significantly reduced compared with that of nonsurgical rats (P < 0.0001 compared with control rats). In addition, cell densities of the ganglion cell layer (GCL), INL, and ONL in vehicle-treated rats (15%, 56%, and 78% that of nonsurgical rats, respectively) were reduced compared with nonsurgical rats (P < 0.0001, P < 0.0001, and P = 0.021 compared with control rats; Fig. 7C ). Destruction of retinal structure was ameliorated significantly in platelet-depleted rats. Thicknesses of the IPL, INL, and ILM-OLM in platelet-depleted rats were 78%, 88%, and 83% (P < 0.0001, P = 0.003, and P < 0.0001 compared with vehicle-treated rats), and cell densities of the GCL, INL, and ONL were 41% and 86% (P = 0.017, P = 0.002, and P = 0.415 compared with vehicle-treated rats). 
Discussion
To address the role of platelets in leukocyte–endothelial cell interactions after ischemia-reperfusion, we used AO and CFSE digital fluorography with an SLO, which allowed us to visualize retinal microcirculation directly and to quantify leukocyte- and platelet-vessel wall interactions with minimal invasion. In the present study, leukocyte rolling along the major retinal veins was dramatically inhibited in platelet-depleted rats during the course of ischemia-reperfusion, and the subsequent leukocyte accumulation in the retinal microcirculation was also significantly suppressed. The expression of P-selectin mRNA was not significantly suppressed in the platelet-depleted postischemia retina, whereas the immunostaining experiment demonstrated significant suppression of P-selectin expression on the retinal vein endothelium. Moreover, intense immunoreactivity was seen in the vascular wall of the vehicle-treated postischemia retinal sections incubated with anti-platelet antibody, which indicates that the suppression of P-selectin immunostaining in platelet-depleted postischemia retina results from the absence of P-selectin expressed on the platelet surface. In addition, administration of GRGDSP inhibited both leukocyte and platelet interactions with vascular endothelium. Taken together, these results strongly suggest that platelets adhering to vascular endothelium contribute to the recruitment of leukocytes through the expression of P-selectin on their surface and participate in leukocyte-dependent tissue injury. 
The contribution of leukocyte infiltration to postischemia damage has been demonstrated by many experimental studies that have demonstrated reduced tissue damage after leukocyte depletion or blockage of adhesion molecules. 1 35 36 Our previous studies showed that the number of accumulated leukocytes in the retinal microcirculation increased significantly, and reached a peak at 24 hours after ischemia-reperfusion. 29 Leukocytes that infiltrate postischemic tissues have been implicated as key mediators of ischemia–reperfusion injury, because they generate oxidants and release proteases. 37 38 Leukocyte infiltration from the mainstream circulation into postischemia tissue is mediated through a multistep process. 5 6 7 In the first step, leukocytes are tethered and roll along the vascular endothelial surface. This phenomenon is reportedly mediated primarily by endothelial P-selectin and carbohydrate ligands of leukocytes. 8 9 Meanwhile, some rolling leukocytes adhere firmly to the vascular endothelium through β2 integrins and ICAM-1, which leads to emigration out of the vasculature. 10 11 We have recently shown that neutralization of P-selectin or ICAM-1 attenuates the postischemia retinal damage 7 and that P-selectin mRNA expression in rat retina is upregulated at 9 to 24 hours after ischemia-reperfusion. Similarly, in another in vivo study, Suzuki et al. 39 40 showed that the P-selectin immunoreactivity begins to be expressed in the microvasculature of the cerebral cortex at 2 hours after reperfusion, and that the expression reaches a maximum at 8 hours to 1 day after reperfusion. This long-term expression of P-selectin, unlike that in other organs, may be due to specificity of the central nervous system. 
In many previous studies, platelets have been shown to reveal active interactions with a subendothelial matrix of injured vessel wall, thereby minimizing loss of blood. 41 42 43 Several glycoproteins, including collagen, vWF, thrombospondin, and fibronectin, have been investigated in attempts to mediate these interactions. 44 45 46 47 This flow-dependent platelet adhesion to a subendothelial matrix leads to rapid platelet aggregation, activation, and P-selectin expression on the platelet surface, which supports leukocyte adhesion under flow conditions. 48 In addition to this involvement in thrombus formation, some investigations suggest that platelets contribute significantly to the inflammatory processes. 20 21 22 Under physiologic conditions, nitric oxide, and prostaglandin I2 derived from vascular endothelial cells provide a nonadhesive vascular surface and prevent platelet-endothelial cell interactions. 49 50 Once endothelial cells are activated, however, they show intermittent adhesion with platelets flowing in the marginal blood flow. 23 31 Meanwhile, platelets reveal stable adhesion to endothelial cells by activation of adhesion molecules on their surfaces, and platelets that accumulate in ischemic regions release oxidants and inflammatory mediators 17 18 19 In our previous studies, 31 many platelets were observed rolling and adhering along the major retinal veins after transient retinal ischemia. We also reported that these interactions are mediated through P-selectin expressed on the retinal endothelial cells in the postischemia retina, whereas P-selectin expressed by α-granules on the platelets plays a minor role in these interactions. Moreover, we demonstrated that the number of adherent platelets along the venous wall substantially increased at 4 hours, reached a peak at 12 hours, and decreased almost to the basal level at 48 hours after reperfusion. It should be noted that the time course of platelet adhesion and leukocyte rolling were parallel and that P-selectin expressed on platelets that that adhere to the postischemia vessel wall mediated leukocyte–endothelial cell interactions. 
Our data suggest an important role for platelets in leukocyte recruitment into postischemia retina. Our previous report indicated that the blockage of P-selectin expression on the retinal vascular wall decreased leukocyte rolling and the subsequent accumulation in retinal microcirculation. In this study, AO digital fluorography demonstrated a significant reduction in leukocyte rolling in platelet-depleted retina after reperfusion, whereas platelet depletion did not alter the expression of P-selectin mRNA in retina. In addition, immunohistologic examination demonstrated the reduction of P-selectin expression on the retinal vascular wall in platelet-depleted postischemia retina. Because activated platelets, similar to activated endothelial cells, express P-selectin on their surfaces from α-granules, we investigated whether platelets would adhere to postischemia vascular wall after blood cells were washed out of the vessels. We observed intense immunoreactivity along the vein wall and noted that leukocytes were adherent to it. Taken together, adherent platelets along the vascular wall after reperfusion are thought to support leukocyte interactions with endothelium and to take part in tissue injury after reperfusion. 
To confirm the role of platelets adhering to the vein wall during reperfusion, the effect of antiadhesive RGD peptide that binds to a variety of integrins, including αvβ3 integrin and GP IIb/IIIa, on platelet- and leukocyte–endothelial interactions was studied. GP IIb/IIIa is the most prominent platelet adhesion receptor, which interacts with several adhesive ligands including fibrinogen, vWF, fibronectin, and vitronectin. The interaction between these ligands and GPIIb/IIIa involves several regions of the ligand, including the motif RGD-peptide. Moreover, Bombeli et al. 24 showed the involvement of the endothelial cell receptor αvβ3 integrin in the binding of platelet-bound fibrinogen. The inhibition of endothelial αvβ3 integrin and fibrinogen interaction by GRGDSP may partially contribute to the reduction of platelet adhesion, leading to the inhibition of leukocyte rolling. In our study, administration of GRGDSP before reperfusion suppressed both platelet adhesion and leukocyte rolling along postischemia retinal veins. These results support the hypothesis that leukocyte recruitment to inflamed endothelium is mediated by adhesion molecules expressed on platelets adherent to endothelial cells. 
In conclusion, we have demonstrated in vivo that platelet depletion suppresses leukocyte rolling and their subsequent accumulation in postischemic tissues. Platelets that adhere to the vascular wall can express P-selectin on their surfaces. This study provides evidence that platelets play a major role in vivo in the recruitment of leukocytes into tissues after ischemia-reperfusion through the expression of adhesion molecules. Antiplatelet therapy may thus ameliorate neural damage after ischemia–reperfusion injury through the control of leukocyte recruitment into tissues after ischemia. 
 
Figure 1.
 
Time course of platelets counts in peripheral blood. *P < 0.0001, †P < 0.001 compared with counts in control rats.
Figure 1.
 
Time course of platelets counts in peripheral blood. *P < 0.0001, †P < 0.001 compared with counts in control rats.
Figure 2.
 
(A) Sequence of fluorescent fundus images of postischemia retina after administration of AO. Arrows: rolling leukocyte along a major retinal vein; arrowheads: adherent leukocyte. (B) Time course of flux of rolling leukocytes after reperfusion in platelet-depleted and vehicle-treated rats. Leukocyte rolling was inhibited significantly in platelet-depleted rats. *P < 0.01, compared with values in vehicle-treated rats. (C) Fundus images with AO digital fluorography of control and postischemia retinas. Leukocytes accumulated in retinal microcirculation appeared as fluorescent dots at 30 minutes after AO injection. Control rat, vehicle-treated rat at 12 and 24 hours after reperfusion (I/R), and platelet-depleted (PD+I/R) rat at 12 and 24 hours after reperfusion. (D) The number of accumulated leukocytes in the postischemia retina of platelet-depleted rats was suppressed significantly. Data are expressed as the mean ± SEM; *P < 0.05 †P < 0.01 compared with results in vehicle-treated rats.
Figure 2.
 
(A) Sequence of fluorescent fundus images of postischemia retina after administration of AO. Arrows: rolling leukocyte along a major retinal vein; arrowheads: adherent leukocyte. (B) Time course of flux of rolling leukocytes after reperfusion in platelet-depleted and vehicle-treated rats. Leukocyte rolling was inhibited significantly in platelet-depleted rats. *P < 0.01, compared with values in vehicle-treated rats. (C) Fundus images with AO digital fluorography of control and postischemia retinas. Leukocytes accumulated in retinal microcirculation appeared as fluorescent dots at 30 minutes after AO injection. Control rat, vehicle-treated rat at 12 and 24 hours after reperfusion (I/R), and platelet-depleted (PD+I/R) rat at 12 and 24 hours after reperfusion. (D) The number of accumulated leukocytes in the postischemia retina of platelet-depleted rats was suppressed significantly. Data are expressed as the mean ± SEM; *P < 0.05 †P < 0.01 compared with results in vehicle-treated rats.
Table 1.
 
Mean Arterial Blood Pressure and Peripheral Blood Leukocyte Count of Rats Treated at the Time of Reperfusion
Table 1.
 
Mean Arterial Blood Pressure and Peripheral Blood Leukocyte Count of Rats Treated at the Time of Reperfusion
Control 4 h 12 h 24 h
Untreated
 MABP (mm Hg) 118.7 ± 4.0 109.3 ± 3.9 116.2 ± 9.5 112.2 ± 6.1
 WBC (×103/μL) 5.0 ± 0.2 9.7 ± 1.8 13.0 ± 1.5 8.1 ± 0.7
 Platelet (×103/μL) 113.1 ± 7.5 102.9 ± 7.6 99.0 ± 1.9 103.2 ± 6.7
Platelet-depleted
 MABP (mm Hg) 118.7 ± 0.8 99.9 ± 8.6 97.5 ± 2.5
 WBC (×103/μL) 6.1 ± 1.1 8.9 ± 1.5 7.5 ± 0.7
 Platelet (×103/μL) 2.6 ± 0.9 3.0 ± 1.0 8.5 ± 1.5
Figure 3.
 
The number of rolling (A) and adherent (B) platelets reached a peak 12 hours after reperfusion. *P < 0.01, †P < 0.05 compared with the control.
Figure 3.
 
The number of rolling (A) and adherent (B) platelets reached a peak 12 hours after reperfusion. *P < 0.01, †P < 0.05 compared with the control.
Figure 4.
 
Inhibitory effects of ARP2-4 and GRGDSP on platelet rolling (A), platelet adhesion (B), and leukocyte rolling (C) 12 hours after ischemia reperfusion. Data are expressed as the mean ± SEM; *P < 0.05 and †P < 0.01 compared with the control.
Figure 4.
 
Inhibitory effects of ARP2-4 and GRGDSP on platelet rolling (A), platelet adhesion (B), and leukocyte rolling (C) 12 hours after ischemia reperfusion. Data are expressed as the mean ± SEM; *P < 0.05 and †P < 0.01 compared with the control.
Figure 5.
 
Immunostaining of frozen sections of retina for vWF and P-selectin. On sections without primary antibody, no immunoreactivity was seen in the venous endothelium. Arrows: venous staining. Compared with the vehicle-treated retina, P-selectin immunoreactivity was decreased in platelet-depleted retina. Bar, 50 μm. Original magnification, ×400.
Figure 5.
 
Immunostaining of frozen sections of retina for vWF and P-selectin. On sections without primary antibody, no immunoreactivity was seen in the venous endothelium. Arrows: venous staining. Compared with the vehicle-treated retina, P-selectin immunoreactivity was decreased in platelet-depleted retina. Bar, 50 μm. Original magnification, ×400.
Figure 6.
 
(A) Immunostaining of retinal sections for platelets. Intense staining suggests that platelets were adhering along vascular walls. (B) An erythrocyte (arrowhead) and a leukocyte (arrow) sticking to the vessel wall.
Figure 6.
 
(A) Immunostaining of retinal sections for platelets. Intense staining suggests that platelets were adhering along vascular walls. (B) An erythrocyte (arrowhead) and a leukocyte (arrow) sticking to the vessel wall.
Figure 7.
 
(A) Light micrographs of retina at a distance of 1.5 mm from the center of the optic nerve head. Nonsurgical control rat retina. Vehicle-treated and platelet-depleted retina at 14 days after reperfusion. Retinal damage was most apparent in the inner retina. Original magnification, ×400. (B) Thickness of various retinal layers in postischemic eyes at 14 days after reperfusion. Data are the mean ± SEM; *P < 0.0001 and §P < 0.05 compared with control rats. †P < 0.01 compared with vehicle-treated rats. (C) Cell density of each retinal layer at 14 days after reperfusion in vehicle-treated and platelet-depleted rats. Data are expressed as the mean ± SEM; *P < 0.01, §P < 0.05 compared with control rats; †P < 0.05, ‡P < 0.01 compared with vehicle-treated rats.
Figure 7.
 
(A) Light micrographs of retina at a distance of 1.5 mm from the center of the optic nerve head. Nonsurgical control rat retina. Vehicle-treated and platelet-depleted retina at 14 days after reperfusion. Retinal damage was most apparent in the inner retina. Original magnification, ×400. (B) Thickness of various retinal layers in postischemic eyes at 14 days after reperfusion. Data are the mean ± SEM; *P < 0.0001 and §P < 0.05 compared with control rats. †P < 0.01 compared with vehicle-treated rats. (C) Cell density of each retinal layer at 14 days after reperfusion in vehicle-treated and platelet-depleted rats. Data are expressed as the mean ± SEM; *P < 0.01, §P < 0.05 compared with control rats; †P < 0.05, ‡P < 0.01 compared with vehicle-treated rats.
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Figure 1.
 
Time course of platelets counts in peripheral blood. *P < 0.0001, †P < 0.001 compared with counts in control rats.
Figure 1.
 
Time course of platelets counts in peripheral blood. *P < 0.0001, †P < 0.001 compared with counts in control rats.
Figure 2.
 
(A) Sequence of fluorescent fundus images of postischemia retina after administration of AO. Arrows: rolling leukocyte along a major retinal vein; arrowheads: adherent leukocyte. (B) Time course of flux of rolling leukocytes after reperfusion in platelet-depleted and vehicle-treated rats. Leukocyte rolling was inhibited significantly in platelet-depleted rats. *P < 0.01, compared with values in vehicle-treated rats. (C) Fundus images with AO digital fluorography of control and postischemia retinas. Leukocytes accumulated in retinal microcirculation appeared as fluorescent dots at 30 minutes after AO injection. Control rat, vehicle-treated rat at 12 and 24 hours after reperfusion (I/R), and platelet-depleted (PD+I/R) rat at 12 and 24 hours after reperfusion. (D) The number of accumulated leukocytes in the postischemia retina of platelet-depleted rats was suppressed significantly. Data are expressed as the mean ± SEM; *P < 0.05 †P < 0.01 compared with results in vehicle-treated rats.
Figure 2.
 
(A) Sequence of fluorescent fundus images of postischemia retina after administration of AO. Arrows: rolling leukocyte along a major retinal vein; arrowheads: adherent leukocyte. (B) Time course of flux of rolling leukocytes after reperfusion in platelet-depleted and vehicle-treated rats. Leukocyte rolling was inhibited significantly in platelet-depleted rats. *P < 0.01, compared with values in vehicle-treated rats. (C) Fundus images with AO digital fluorography of control and postischemia retinas. Leukocytes accumulated in retinal microcirculation appeared as fluorescent dots at 30 minutes after AO injection. Control rat, vehicle-treated rat at 12 and 24 hours after reperfusion (I/R), and platelet-depleted (PD+I/R) rat at 12 and 24 hours after reperfusion. (D) The number of accumulated leukocytes in the postischemia retina of platelet-depleted rats was suppressed significantly. Data are expressed as the mean ± SEM; *P < 0.05 †P < 0.01 compared with results in vehicle-treated rats.
Figure 3.
 
The number of rolling (A) and adherent (B) platelets reached a peak 12 hours after reperfusion. *P < 0.01, †P < 0.05 compared with the control.
Figure 3.
 
The number of rolling (A) and adherent (B) platelets reached a peak 12 hours after reperfusion. *P < 0.01, †P < 0.05 compared with the control.
Figure 4.
 
Inhibitory effects of ARP2-4 and GRGDSP on platelet rolling (A), platelet adhesion (B), and leukocyte rolling (C) 12 hours after ischemia reperfusion. Data are expressed as the mean ± SEM; *P < 0.05 and †P < 0.01 compared with the control.
Figure 4.
 
Inhibitory effects of ARP2-4 and GRGDSP on platelet rolling (A), platelet adhesion (B), and leukocyte rolling (C) 12 hours after ischemia reperfusion. Data are expressed as the mean ± SEM; *P < 0.05 and †P < 0.01 compared with the control.
Figure 5.
 
Immunostaining of frozen sections of retina for vWF and P-selectin. On sections without primary antibody, no immunoreactivity was seen in the venous endothelium. Arrows: venous staining. Compared with the vehicle-treated retina, P-selectin immunoreactivity was decreased in platelet-depleted retina. Bar, 50 μm. Original magnification, ×400.
Figure 5.
 
Immunostaining of frozen sections of retina for vWF and P-selectin. On sections without primary antibody, no immunoreactivity was seen in the venous endothelium. Arrows: venous staining. Compared with the vehicle-treated retina, P-selectin immunoreactivity was decreased in platelet-depleted retina. Bar, 50 μm. Original magnification, ×400.
Figure 6.
 
(A) Immunostaining of retinal sections for platelets. Intense staining suggests that platelets were adhering along vascular walls. (B) An erythrocyte (arrowhead) and a leukocyte (arrow) sticking to the vessel wall.
Figure 6.
 
(A) Immunostaining of retinal sections for platelets. Intense staining suggests that platelets were adhering along vascular walls. (B) An erythrocyte (arrowhead) and a leukocyte (arrow) sticking to the vessel wall.
Figure 7.
 
(A) Light micrographs of retina at a distance of 1.5 mm from the center of the optic nerve head. Nonsurgical control rat retina. Vehicle-treated and platelet-depleted retina at 14 days after reperfusion. Retinal damage was most apparent in the inner retina. Original magnification, ×400. (B) Thickness of various retinal layers in postischemic eyes at 14 days after reperfusion. Data are the mean ± SEM; *P < 0.0001 and §P < 0.05 compared with control rats. †P < 0.01 compared with vehicle-treated rats. (C) Cell density of each retinal layer at 14 days after reperfusion in vehicle-treated and platelet-depleted rats. Data are expressed as the mean ± SEM; *P < 0.01, §P < 0.05 compared with control rats; †P < 0.05, ‡P < 0.01 compared with vehicle-treated rats.
Figure 7.
 
(A) Light micrographs of retina at a distance of 1.5 mm from the center of the optic nerve head. Nonsurgical control rat retina. Vehicle-treated and platelet-depleted retina at 14 days after reperfusion. Retinal damage was most apparent in the inner retina. Original magnification, ×400. (B) Thickness of various retinal layers in postischemic eyes at 14 days after reperfusion. Data are the mean ± SEM; *P < 0.0001 and §P < 0.05 compared with control rats. †P < 0.01 compared with vehicle-treated rats. (C) Cell density of each retinal layer at 14 days after reperfusion in vehicle-treated and platelet-depleted rats. Data are expressed as the mean ± SEM; *P < 0.01, §P < 0.05 compared with control rats; †P < 0.05, ‡P < 0.01 compared with vehicle-treated rats.
Table 1.
 
Mean Arterial Blood Pressure and Peripheral Blood Leukocyte Count of Rats Treated at the Time of Reperfusion
Table 1.
 
Mean Arterial Blood Pressure and Peripheral Blood Leukocyte Count of Rats Treated at the Time of Reperfusion
Control 4 h 12 h 24 h
Untreated
 MABP (mm Hg) 118.7 ± 4.0 109.3 ± 3.9 116.2 ± 9.5 112.2 ± 6.1
 WBC (×103/μL) 5.0 ± 0.2 9.7 ± 1.8 13.0 ± 1.5 8.1 ± 0.7
 Platelet (×103/μL) 113.1 ± 7.5 102.9 ± 7.6 99.0 ± 1.9 103.2 ± 6.7
Platelet-depleted
 MABP (mm Hg) 118.7 ± 0.8 99.9 ± 8.6 97.5 ± 2.5
 WBC (×103/μL) 6.1 ± 1.1 8.9 ± 1.5 7.5 ± 0.7
 Platelet (×103/μL) 2.6 ± 0.9 3.0 ± 1.0 8.5 ± 1.5
×
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