In Vivo Evaluation of Leukocyte Dynamics in Retinal
Ischemia Reperfusion Injury
Akitaka Tsujikawa, Yuichiro Ogura, Naoko Hiroshiba, Kazuaki Miyamoto, Junichi Kiryu,
and Yoshihito Honda
evaluate quantitatively leukocyte dynamics in vivo in the rat retinal microcirculation
during ischemia reperfusion injury with the use of acridine orange digital fluorography.
PURPOSE. TO
METHODS. Retinal
ischemia was induced in anesthetized pigmented rats by a temporary ligation of
the optic nerve. After 60 minutes of ischemia, leukocyte behavior in the retinal microcirculation
was evaluated, with acridine orange digitalfluorography—consisting of a scanning laser ophthalmoscope and the fluorescent nuclear dye, acridine orange—during reperfusion at 1, 2, 4, 6, 12, 24,
48, 96, and 168 hours. The obtained images were recorded on videotape and analyzed with a
computer-assisted image analysis system.
RESULTS. Rolling
leukocytes along the major retinal veins were observed in treated rats during the
reperfusion period; no rolling leukocytes were observed in the control rats. The number of rolling
leukocytes gradually increased and peaked at 102 ± 40 cells/minute 12 hours after reperfusion; few
rolling leukocytes were observed at 96 hours. The velocity of rolling leukocytes at 12 hours (19.1 ±
35 /xm/second; P < 0.05) was significantly lower than that at the other three times. No rolling
leukocytes were observed along the arterial walls throughout the experiments. The number of
accumulated leukocytes increased as time elapsed, peaked at 931 ± 187 cells/mm2 24 hours after
reperfusion, and decreased thereafter.
Leukocyte dynamics in the retinal microcirculation can be quantitatively evaluated
during ischemia reperfusion injury. (Invest Ophthalmol Vis Sci. 1998;39:793-800)
CONCLUSIONS.
schemia reperfusion injury has been reported in a variety of
clinical1 and experimental conditions, including myocardial infarction12; stroke3'4; and mesenteric,5 renal,6 and
peripheral vascular disease.7 In findings in many studies, leukocytes have been shown to accumulate in tissue after ischemia2'4 ~'° and to cause tissue injury by blocking blood
flow"12 or by producing the superoxide anion radical,1314
hydrogen peroxide.' In results of early experimental studies, it
has been shown that leukocyte depletion greatly attenuates
postischemic cellular dysfunction in various organs.2'3'7 Results in subsequent studies have demonstrated that the extent
of reperfusion-induced tissue damage is reduced by agents that
prevent leukocyte activation or accumulation.51516 Therefore,
it is suggested that leukocytes play a central role in ischemia
reperfusion injury.2"4'910 The recruitment of circulating leukocytes into tissue after ischemia initially requires interaction
between microvascular endothelial cells and these leukocytes
through specific adhesion molecules.910 Recently, advances
I
From the Department of Ophthalmology and Visual Sciences,
Kyoto University Graduate School of Medicine, Japan.
Supported by a grant for scientific research from the Ministry of
Education, Science, and Culture (YO, JK, YH) and a grant for scientific
research from the Ministry of Health and Welfare of Japan QK).
Presented in part at the Association for Research in Vision and
Ophthalmology annual meeting, Fort Lauderdale, Florida, May 1997.
Submitted for publication May 5, 1997; revised September 3, 1997
and November 15, 1997; accepted December 18, 1997.
Proprietary interest category: N.
Reprint requests: Yuichiro Ogura, Department of Ophthalmology
and Visual Sciences, Kyoto University Graduate School of Medicine,
Sakyo-ku, Kyoto 606, Japan.
have been made in the understanding of the mechanisms of
leukocyte adhesion and migration.91017
Because the retina belongs to the central nervous system
and is vulnerable to ischemia, retinal ischemia reperfusion
injury has been studied by many investigators. 8121318 " 20 Although leukocytes may play an important role in ischemia
reperfusion injury in the retina, leukocyte behavior in the
retina after ischemia has been reported in a only few studies. 8 1 2 1 3
Acridine orange digital fluorography,21"25 which we developed recently, allows visualization of leukocytes and quantitative evaluation of leukocyte dynamics in the retinal microcirculation in vivo. In this technique, a scanning laser
ophthalmoscope, coupled with a computer-assisted image
analysis system, makes continuous high-resolution images of
leukocytes stained by a metachromatic fluorochrome of acridine orange.26 The purpose of this study was to use acridine
orange digital fluorography to evaluate leukocyte dynamics
quantitatively in retinal ischemia reperfusion injury in vivo.
METHODS
Animal Model
Transient retinal ischemia was induced according to the
method described by Stefansson et al.,20 with slight modification. Male pigmented Long Evans rats (200-250 g; n = 65)
were anesthetized with a mixture (1:1) of 4 mg/kg xylazine
hydrochloride and 10 mg/kg ketamine hydrochloride. The pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride. After a lateral conjunctival peritomy and
Investigative Ophthalmology & Visual Science, April 1998, Vol. 39, No. 5
Copyright © Association for Research in Vision and Ophthalmology
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1. Digitized monochromatic images of major retinal vessels obtained with scanning laser ophthalmoscope in the
nonsurgical control rat group (A) and in the operation group 4 (B), 24 (C), and 96 (D) hours after reperfusion. (B) Arteries and
veins showed significant vasoconstriction immediately after reperfusion, with the peak occurring 4 hours afterward, with marked
loss of nerve fiber layer visualization because of significant edema, (C) Subsequent vasodilation peaked 12 to 24 hours after
reperfusion. (D) Vasodilation subsided, and a marked loss of nerve fiber layer was observed 96 hours after reperfusion.
FIGURE
disinsertion of the lateral rectus muscle, the optic nerve of the
right eye was exposed by blunt dissection. A 6-0 nylon suture
was passed around the optic nerve and was tightened until
blood flow ceased in all of the retinal vessels. Complete nonperfusion was confirmed through an operating microscope and
maintained for 60 minutes. Thereafter, nonperfusion was confirmed through an operating microscope, and the suture was
removed. Reperfusion of the vessels was observed through the
operating microscope. Eyes that failed to reperfuse within 5
minutes were excluded from the experiment. Rats in the shamoperation group underwent similar surgery but without tightening of the suture. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
and cytochemical studies.26'27 The dye emits a green fluorescence when it interacts with doubte-stranded DNA. The spectral properties of acridine orange-DNA complexes are similar
to those of sodium fluorescein, with an excitation maximum at
502 nm and an emission maximum at 522 nm.26 The argon
blue laser was used for the illumination source, with a regular
emission filter normally used in fluorescein angiography. Immediately after acridine orange solution was infused intravenously, leukocytes were stained selectively among circulating
blood cells. Nuclei of vascular endothelial cells also were
stained. The obtained images were recorded on an S-VHS
videotape at the video rate of 30 frames/second for further
analysis.
Experimental Design
Acridine Orange Digital Fluorography
Acridine orange digital fluorography has been described elsewhere. 21 ' 25 In this technique, a scanning laser ophthalmoscope (Rodenstock Instruments, Munich, Germany) coupled
with a computer-assisted image analysis system makes continuous high-resolution images of fundus stained by the nietachromatic fluorochrome, acridine orange26 (Wako Pure Chemicals,
Osaka, Japan). Acridine orange is widely used in biochemical
Acridine orange digital fluorography was performed 1, 2, 4, 6,
12, 24, 48, 96, and 168 hours after reperfusion. In the shamoperation group, the examination was performed 4, 12, and 24
hours after surgery. Rats that did not undergo surgery were
evaluated as a control. Five rats were examined at each time
point after surgery. In preliminary experiments, arterial blood
pressure was monitored by the blood pressure analyzer (model
179; IITC, Woodland Hill, CA). Mean arterial blood pressures
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IOVS, April 1998, Vol. 39, No. 5
Leukocyte Dynamics in Retinal Ischemia Reperfusion
were 93 — 15 mm Hg in the control group, and 86 ± 14 mm
Hg and 102 ± 10 mm Hg at 12 and 24 hours, respectively, after
reperfusion in the operation group. No depression of arterial
blood pressure was recognized.
Immediately before acridine orange digital fluorography,
rats were anesthetized with the same agent, and the pupils
were dilated. 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.
Acridine orange (0.1% solution in saline) was injected continuously throvigh the catheter for 1 minute at a rate of 1 ml/
minute. The fundus was observed with the scanning laser
ophthalmoscope in the 40° field for 5 minutes. Thirty minutes
after the injection of acridine orange, the fundus was observed
again to evaluate leukocytes accumulated in the retinal microcirculation.
After the experiment, the rat was killed with an anesthetic
overdose, and the eye was enucleated to determine a calibration factor with which to convert values measured on a computer monitor (in pixels) into real values (in micrometers). The
calibration factor is the ratio between the actual size of each
optic disc measured using a microscope and the apparent value
on a computer monitor.
60
r
50
40
30
20
10
vein
artery
control
Image Analysis
The video recordings were analyzed with an image analysis
system, which has been described in detail elsewhere.21'22 In
brief, the system consists of a computer equipped with a video
digitizer (Radius, San Jose, CA), which digitizes the video image
in real time (30 frames/second) to 640 horizontal and 480
vertical pixels, with an intensity resolution of 256 steps. To
investigate the leukocyte dynamics in the microcirculation of
the retina after ischemia, the investigators evaluated diameters
of major retinal vessels, thefluxof rolling leukocytes along the
major retinal veins, the velocity of rolling leukocytes, and the
number of leukocytes accumulated in the retinal microcirculation through the use of this system.25
Diameters of major retinal vessels were measured at 1 disc
diameter from the center of the optic disc in monochromatic
images recorded before acridine orange injection. Each vessel
diameter was calculated in pixels as the distance between the
half-height points determined separately on each side of the
density profile of the vessel image. The obtained data were
converted into real values by using the calibration factor mentioned above. The averages of the individual arterial and venous diameters were used as the arterial and venous diameters
for each rat.
Rolling leukocytes were defined as leukocytes that move
at a lower velocity than free-flowing leukocytes. The number of
rolling leukocytes was calculated from the number of cells
crossing a fixed area of the vessel at a distance 1 disc diameter
from the optic disc center per minute. The flux of rolling
leukocytes was defined as the total number of rolling leukocytes along all major veins (cells per minute). The velocity of
rolling leukocytes was calculated as the time required for a
leukocyte to travel a given distance (25 pixels) along the vessel.
The average of at least 10 velocities was defined as the velocity
of rolling leukocytes for each rat.
The number of leukocytes accumulated in the retinal
microcirculation was evaluated 30 minutes after acridine
orange injection. The number of fluorescent dots in the
retina within 8 to 10 areas of 100 pixels square at a distance
795
12 24
1
sham-operation
2
4
6
12 24 48 96 168
operation
Reperfusion period (hours)
2. Time course showing changes in major retinal arterial and venous diameters after reperfusion. Values are
mean ± SD. *P < 0.01, \P < 0.05, compared with values in the
sham-operation group; %P < 0.01, §P < 0.05 compared with
values in the surgical group 4 hours after reperfusion.
FIGURE
of 1 disc diameter from the edge of the optic disc was
counted. The average number of individual areas was used
as the number of leukocytes accumulated in the retinal
microcirculation in each rat.
Statistical Analysis
All values are presented as mean ± standard deviation. Analysis
of variance was used to compare three or more conditions,
with posthoc comparisons tested using Fisher's protected least
significant difference (PLSD) test. Differences were considered
statistically significant when P values were less than 0.05.
RESULTS
Diameters of Major Retinal Vessels
In Figures 1 and 2, the time course of major retinal vessel
diameters is shown in the nonsurgical control, sham-operation,
and operation groups at various time points after reperfusion.
In arteries and veins, vasoconstriction occurred immediately
after reperfusion and peaked 4 hours after reperfusion in the
operation group (66.8%; P < 0.01, 90.1%; P < 0.05). Afterward, significant vasodilation occurred in arteries and veins. In
arteries, vasodilation peaked 12 to 24 hours after reperfusion
(123-129%; P < 0.01) compared with the value at 4 hours after
reperfusion and subsided 96 hours after reperfusion. Vasodilation was more remarkable in veins than in arteries. Venous
vasodilation, which reached a peak 24 hours after reperfusion,
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Tsujikawa et al.
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3- A leukocyte observed rolling along the venous wall (arrowhead). Another leukocyte rolled along the wall of a major
retinal vein (closed arrow) and adhered to the venous wall (open arrow).
FIGURE
was more significant (P < 0.05) in the surgical group than in
the sham-operation group, and subsided 48 hours after reperfusion.
Rolling of Leukocytes
Immediately after acridine orange was infused, many freeflowing leukocytes were visualized. Because free-flowing leukocytes in the major retinal vessel move rapidly, it is likely that
the image of a free-flowing leukocyte was nonspherical-that is,
slightly elongated. It could be recognized as a single particle,
however, even if the image was blurred. In some postischemic
conditions, leukocytes were observed rolling along the major
retinal veins, making intermittent adhesive contact with vascular endothelial cells. But no rolling leukocytes were observed
in the major retinal arteries throughout all of the experiments
(Fig. 3).
No rolling leukocytes were seen in the major retinal veins
in the control, sham-operation, or operation group 1 hour after
reperfusion; leukocytes began to roll along the venous walls 4
hours after reperfusion. The flux of rolling leukocytes substantially increased and reached a peak (102 ± 40 cells/minute) 12
hours after reperfusion. The flux decreased notably to approximately one sixth the maximum level 48 hours after reperfusion. Few rolling leukocytes could be observed 96 and 168
hours after reperfusion (Fig. 4).
Velocity of Rolling Leukocytes
The velocities of rolling leukocytes 2, 48, and 168 hours after
reperfusion could not be determined, because insufficiently
low numbers (fewer than 10) of rolling leukocytes were observed during the experiments. Leukocyte rolling velocities in
the operation group 4, 6, and 24 hours after reperfusion were
28.5 ± 3.7 ju.ni/second, 26.5 ± 3-4 /xm/second, and 27.2 ± 35
/jim/second, respectively, and there was no significant difference among them. The velocity of rolling leukocytes 12 hours
after reperfusion, however, was significantly lower (19.1 ± 3.5
/jtm/second; P < 0.05) than that recorded at 4, 6, and 24 hours
(Fig. 5).
Leukocytes Accumulated in the Retinal
Microcirculation
Because acridine orange is membrane permeable, it easily infiltrates through the vessel wall and diffuses into the retina. A
few minutes after acridine orange injection was stopped, circulating leukocytes showed less fluorescence, probably owing
to the washout effect. In contrast, leukocytes that accumulated
in the retina remained fluorescent for approximately 2 hours.
Therefore, leukocytes that accumulated in the retina were
recognized as distinct fluorescent dots 30 minutes after acridine orange injection, although no circulating leukocytes fluoresced (Fig. 6).
In Figure 7 the changes in number of leukocytes accumulated in the retinal microcirculation are indicated. Although
few leukocytes could be recognized in the control and shamoperation groups, accumulated leukocytes began to increase in
the operation group 4 hours after reperfusion. The number
increased with time and peaked at 931 ± 187 cells/mm2 24
hours after reperfusion. The number of accumulated leukocytes decreased after that and remained at approximately one
third the maximum level.
DISCUSSION
In this study, leukocytes were clearly visualized and quantitatively evaluated rolling along the retinal vessel wall and accumulating in the retinal microcirculation after transient retinal
ischemia in vivo, The rolling of leukocytes is the phenomenon
in which leukocytes make intermittent adhesive contact with
vascular endothelial cells28 and is suggested to be thefirststep
in the accumulation of leukocytes in tissue after ischemia. 51017 In the physiologic state, although rolling leukocytes are observed along the venous wall in some organs,16'29
no rolling leukocytes have been observed in the retinal major
veins. Baatz et al.30 have reported that the velocity of freeflowing leukocytes was 2.57 ± 0.07 mm/second in iris venules;
Nishiwaki et al.22 reported that the velocity was 17.4 ± 5.3
mm/second in the major retinal veins. From these reports, the
velocity of free-flowing leukocytes is much faster in the retina
than in the iris. The higher speed of flow in retinal vessels,
compared with that in other vessels, may be why rolling
leukocytes are absent in the normal retina. In a pathologic
state, however, such as that occurring after ischemia, rolling
leukocytes were observed along the venous wall against the
higher flow speed. In this study, the velocity of rolling leukocytes ranged from 7.0 to 59-2 ptm/second (average, 25.4 ± 53
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Leukocyte Dynamics in Retinal Ischemia Reperfusion
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140 r
120
1 100
Iu
80
o>
60
o
40
20
control 4
12 24
sham-operation
1
2
4
6
12 24 48 96 168
operation
Reperfusion period (hours)
4. Time course of flux of rolling leukocytes after
reperfusion. Values are mean ± SD.
FIGURE
/xm/second). Even the fastest rolling leukocyte moved almost
300 times more slowly than the average of free-flowing leukocytes. Therefore, by observing the monitor, it was possible to
see two groups of leukocytes with two apparently different
velocities: free-flowing and rolling leukocytes. Because no leukocytes with intermediate velocity were observed, it was not
difficult to distinguish rolling leukocytes from free-flowing leukocytes.
In Figure 4, the time course of the number of rolling
leukocytes is outlined. In this study, rolling leukocytes were
first observed along the venous walls 4 hours after reperfusion.
The number of rolling leukocytes increased substantially and
reached a peak at 12 hours; they decreased to control levels at
96 hours. The time course in this study was delayed, compared
with that in other organs. It is reported that rolling leukocytes
peak 30 minutes after reperfusion and almost subside at 24
hours in the dorsal skinfold chamber model of hamsters,29 in
mice,16 and in the mesentery of rats.31 This difference may
derive from the specificity of the retina subject to ischemia
reperfusion injury.832
According to recent advances in understanding the mechanisms of leukocyte adhesion and migration, leukocyte rolling
is thought to be mediated by interaction between the selectin
family (P-, E-, and L-selectin) and its counter ligand, sialyl
LewisX.5'28 Okada et al.33 showed significant and persistent
upregulation of P-selectin after focal brain ischemia and reperfusion. Recently, Zhang et al.34 have shown that mRNA expression of E-selectin was upregulated in the cerebrum after transient middle cerebral artery occlusion and peaked 10 hours
after reperfusion. We speculate that upregulation occurred in
the retina after ischemia and that upregulated selectins medi-
797
ated leukocyte rolling along the major retinal veins to accumulate in the retina after ischemia.
In this study, the velocity of rolling leukocytes 12 hours
after reperfusion was significantly lower (19.1 ± 35 jixm/
second; P < 0.05) than the velocities at other times. The
velocity of free-flowing leukocytes was 135 ± 5.1 mm/second
in the control rats and 10.2 ± 2.8 mm/second, 12.2 ± 4
mm/second, 10.4 ± 3 mm/second at 4, 12, and 24 hours after
reperfusion in the operation group. The velocity of free-flowing leukocytes showed no significant changes during the experiment. The difference in kind and density of adhesion molecules involved in leukocyte rolling at each time point may
account for this finding.10'28
Whereas leukocytes make weak adhesive contact with
vascular endothelial cells, they are activated by interaction
with endothelial cells and adhere firmly to them. 910 ' 28 In this
study, accumulated leukocytes began to increase substantially
4 hours after reperfusion and reached a peak level of 931 ±
187 cells/mm2 at 24 hours. In previous studies, Szabo et al.13
reported that leukocyte infiltration to the retina increased after
90 minutes of retinal ischemia and 24 hours of reperfusion; 60
minutes of ischemia and 24 hours of reperfusion did not lead to
migration. Hangai et al.8 showed that neutrophil migration in
the retina subject to ocular ischemia for 2 hours was never
observed before 3 hours after reperfusion; it was readily apparent after 12 hours. Although their observation is partially
compatible with our findings, they may have underestimated
the number of leukocytes. Because they observed accumulated
leukocytes on histologic sections, they could not evaluate all
leukocytes accumulated in the retina after ischemia. Conversely, our allows quantitative evaluation of all leukocytes
accumulated in the retina after ischemia.
35
30
25
20
7?
15
10
4
6
12
Reperfusion period (hours)
5. Velocities of rolling leukocytes at time points after
reperfusion. The leukocytes' rolling velocity at 12 hours was
significantly lower than values at 4, 6, and 24 hours. Values are
mean ± SD. *P < 0.05 compared with the values at 12 hours.
FIGURE
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Tsujikawa et al.
IOVS, April 1998, Vol. 39, No. 5
FIGURE 6.
Leukocytes accumulated in the retinal microcirculation appear as fluorescent dots
30 minutes after acridine orange injection. A small number of leukocytes could be seen in the
control rats (A). Increasing numbers of leukocytes accumulated after reperfusion and peaked
24 hours after reperfusion in the surgical rats (B).
The |32-integrin family (CDlla/CDlS, CDllb/CD18) and
the immunoglobulin superfamily (intercellular adhesion molecule 1 [ICAM-1]), have been suggested as mediators in firm
adhesion between leukocytes and endothelial cells. 4910 ' 35 Although ICAM-1 is constitutively expressed on endothelial
cells,15 Wang et al.35 have shown that mRNA expression of
ICAM-1 is upregulated after transient cerebral ischemia and
peaks 12 hours after reperfusion. Kim et al.36 have recently
shown that CDlla and CD18 are upregulated in leukocytes
from patients who have had ischemic stroke. Our observations
in the retina are consistent with their results in the cerebrum,
and we speculate that similar upregulation occurred in the
retina after ischemia and that these upregulated adhesion molecules mediated the interaction between accumulated leukocytes and retinal vascular endothelial cells.
In this study, as is shown in Figure 1, significant vasoconstriction and subsequent vasodilation occurred in arteries and
veins after reperfusion. Significant vasoconstriction was observed immediately after reperfusion and reached maximum
levels 4 hours after reperfusion. Vascular endothelium is a
source of relaxing (nitric oxide INO]) and contracting factors
(endothelin), and interactions between these factors contribute to the vascular tone.57'38 It is reported that endothelium
injured by ischemia releases insufficient NO after reperfu-
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1200
r
1000
(0
%
800
Q)
600
400
200
control4
12 24
1
sham-operation
2
4
6
12 24 48 96 168
operation
Reperfusion period (hours)
FIGURE 7.
Time course of the number of leukocytes accumulated in the retinal microcirculation. Values are mean ± SD.
sion.39 This imbalance may contribute to marked vasoconstriction after reperfusion.40 On the contrary, Hangai et al.41
showed that mRNA expression of inducible nitric oxide synthase (iNOS) was highly upregulated 12 to 24 hours after
reperfusion, when the retina was subjected to ischemia for 2
hours. They also showed that mRNA of iNOS was expressed in
the resident retinal cells and neutrophils that had infiltrated the
retina. As is shown in Figure 7, leukocytes began to accumulate
in tissue after ischemia and peaked after 24 hours of reperfusion. A large amount of NO produced by accumulated leukocytes may contribute to significant vasodilation in arteries and
veins after vasoconstriction. Hangai et al.44 also reported that
mRNA expression of iNOS returned to normal levels 48 hours
after reperfusion, a finding that is compatible with our results
showing that vasoconstriction was dominant 48 hours after
reperfusion. Accumulated leukocytes do not produce as much
NO 48 hours after reperfusion. .
In conclusion, rolling along the venous wall is essential for
leukocytes to disseminate in tissue after ischemia and to produce tissue injury. 51017 Therefore, investigating leukocyte dynamics in the retina after ischemia is valuable in estimating
ischemia reperfusion injury and in evaluating the protective
effect of drugs. In the present study dynamic behavior of
leukocytes was visualized in the retina after ischemia. The
method used will allow quantification of the severity of ischemia reperfusion injury and quantitative evaluation of the effectiveness of therapies for retinal ischemia.
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