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From the Department of Ophthalmology, Okayama University Medical School, Japan.
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Abstract |
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METHODS. Pigmented rabbits and monkeys were used in this study. Leukocytes, collected by centrifugal separation of autologous blood, were stained with indocyanine green (ICG) dye. The ICG-stained leukocyte fluid was injected into the vein, and the fundus image was obtained with a scanning laser ophthalmoscope. The image was recorded on videotapes and analyzed with a personal computer-based image analysis system.
RESULTS. In pigmented rabbits, fluorescent leukocytes moving in the choroidal circulation were clearly visible for more than 1 hour. In monkeys, distinct fluorescent dots were seen moving approximately 50 to 200 µm in the foveal avascular zone for more than 30 minutes after the injection of the ICG-stained leukocyte fluid. Dim fluorescent dots were seen moving in the fundus. Although the movement of these dim dots was difficult to trace, they seemed to be moving in the choroidal vessels. In the rabbits, the mean flow velocity of leukocytes moving without plugging was 0.48 ± 0.14 mm/sec in the peripheral choriocapillaris. In the monkeys, the mean flow velocity of distinct fluorescent leukocytes without plugging was 2.45 ± 0.48 mm/sec in the posterior choroid.
CONCLUSIONS. In pigmented rabbits and monkeys, this method allows visualization of leukocytes passing through the choroidal vessels and provides a new way to investigate, noninvasively and in vivo, leukocyte dynamics in the choroidal microcirculation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We developed a new method with which to directly visualize leukocytes in the choroid of pigmented animals and investigated leukocyte dynamics in the choroidal circulation of pigmented rabbits and monkeys through autologous leukocytes stained with ICG dye. Our method, called indocyanine green leukocyte angiography (ILA), can be applied to the evaluation of leukocyte dynamics in the choroidal circulation and may be feasible for use in humans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Indocyanine Green Leukocyte Angiography
Ten to 20 ml blood was withdrawn from a vein (in rabbits from the
ear vein; in monkeys from the cubital vein) into a sterile test tube.
The blood was mixed with a mixture of Ficoll and metrizoate (Mono-Poly
Resolving Medium; Dainippon Pharmaceutical, Osaka, Japan) and separated
using a centrifuge at 1800 rpm for 30 minutes. Most of the plasma and
the bulk of erythrocytes were removed and the white-coat layer of
leukocytes was mixed with 0.01 ml (in rabbits) or 0.05 ml (in monkeys)
ICG solution (Diagnogreen injection; Daiich Pharmaceutical, Tokyo,
Japan). Additional centrifugal separation of the leukocyte fluid at
1500 rpm for 5 minutes was performed to collect ICG-stained leukocytes
more densely. Three milliliters of phosphate-buffered saline (PBS) was
added to the ICG-stained leukocytes. The whole leukocyte fluid was
estimated to contain 10 to 60 million leukocytes by counting the
leukocytes in 0.1 µl of the leukocyte fluid.
Pupils were dilated using 0.5% tropicamide ophthalmic solution. The leukocyte fluid was injected into the ear vein (in rabbits) or the antecubital vein (in monkeys), and the fundus images were obtained with infrared laser and an SLO (model 101; Rodenstock Instrument, Munich, Germany). The SLO was operated using a 40° field size in rabbits or a 20° field size in monkeys. The images, obtained at 30 frames/sec, were recorded on S-VHS videotapes. Twenty minutes after the injection, simultaneous fluorescein and ICG angiography,20 showing retinal vascular landmarks, was performed to locate ICG-stained leukocytes in the monkeys.
Image Analysis
Images were analyzed with a computer-based image analysis system.
Consecutive images were captured with an analog–digital converter
board (Dig98; Ditect, Tokyo, Japan) loaded into a personal computer
(PC9821Xa; NEC, Tokyo, Japan). Consecutive images were superimposed
using original software running on Windows 95 (Microsoft Japan, Tokyo,
Japan) so that the movement of fluorescent dots could be traced on one
image. The center portion of leukocytes was marked manually on a
computer monitor, and the distance between consecutively marked
positions was measured in pixels as a spline curve,5
which
has been applied to tracer flow extraction in arterial blood flow
studies. The flow velocity, calculated in pixels from the distance of
consecutive portions, was converted into a real value with the aid of a
calibration factor determined in an optical realistic model of the
human eye for the monkey experiment.6
For the rabbit, a
real size was measured in vivo after insertion of a thin ruler on the
surface of the retina using the SLO. The velocity of 10 leukocytes
moving in the choroidal vessels was measured in each animal.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pigmented Rabbits
Composite photographs of early-phase images and late-phase images
were used for the differentiation between arteries and veins. Many
fluorescent dots became distinctly visible approximately 5 minutes
after injection. For more than 1 hour, each ICG-stained leukocyte
passing through the choroidal circulation was distinctly observed as a
single fluorescent dot moving in the choroidal vessels. Because
choroidal arteries and veins were outlined as negative figures against
the background, it was possible to observe these leukocytes moving
rapidly in the choroidal arteries, passing very slowly through the
choroidal capillaries and draining into the choroidal veins at
increasing velocity (Fig. 1)
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The flow velocities of the fluorescent leukocytes that did not plug decreased rapidly at the entry point of the choroidal capillaries and were constant during passage through the capillaries. The flow velocities of leukocytes that did not plug, which passed through the peripheral capillaries, ranged from 0.26 to 0.93 mm/sec (mean velocity ± SD, 0.48 ± 0.14 mm/sec).
Monkeys
Approximately 10 minutes after the injection, fluorescent dots
became evident in the posterior region, and afterward each leukocyte
was distinctly recognized as a single fluorescent dot in the posterior
region for more than 30 minutes by using a 20° field size. These
fluorescent leukocytes were located with retinal vascular landmarks and
negative images of the choroidal vessels.
In the monkey, there were two patterns of leukocytic movement. In one pattern, fluorescent dots moved from the retinal artery toward the perifoveal network, passed through the perifoveal capillaries, and drained into the retinal veins (Fig. 2) . It was possible to consecutively trace the movement of the fluorescent dots moving through the retinal circulation. The mean flow velocity of leukocytes passing through the perifoveal retinal capillaries was 1.10 ± 0.37 mm/sec. In the other pattern, fluorescent dots appeared abruptly on a video monitor, moved approximately 50 to 200 µm, and disappeared abruptly (Fig. 3) . Because the latter pattern was often observed in the foveal avascular zone (Fig. 4) , the fluorescent dots moving in this pattern represent ICG-stained leukocytes in the choroidal circulation. The mean flow velocity passing the choroidal vessels was 2.45 ± 0.48 mm/sec. This was calculated based on two or three consecutive images by which the movement of each dot could be traced. In both patterns, transient plugging was sometimes observed. Additionally, dim fluorescent dots were observed moving too fast to be recognized in the still image. Although the movement of these dim dots was difficult to trace, it was substantially different from that of bright dots moving in the retinal circulation. These dots seemed to be moving in the choroidal vessels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The flow velocity of leukocytes moving in the choroidal vessels was measured in the present study. The fastest velocity that could be measured by using the viewing system in the present study was estimated to be 30 mm/sec. In rabbits, fluorescent dots in the choroidal arteries were clearly observed. However, the movement in the arteries was too fast to be traced on a monitor, and it was difficult to evaluate the velocity of them accurately. Koyama et al.7 reported that the flow velocity of erythrocytes moving in the choriocapillaris of albino rabbits, which was measured by using high-speed videography taken through a scleral window, ranged from 0.28 to 2.11 mm/sec and that leukocytes moved more slowly than erythrocytes. Matsuda et al.2 reported that leukocyte velocity in the choroidal capillaries was 0.74 ± 0.06 mm/sec in the nonpigmented rats. In the present study, the mean velocity was 0.48 ± 0.14 mm/sec. The discrepancy may be due to the difference in species or measurement sites.
In primates, little is known about leukocyte rheology in the choroidal circulation, and the flow velocity of leukocytes in the choroid is unknown, because there is no available method. In contrast, the mean flow velocity of leukocytes passing through the retinal perifoveal capillaries was reported to be 0.92 ± 0.32 mm/sec in monkeys1 and 1.41 ± 0.29 or 0.54 ± 0.19 mm/sec in humans.4 8 These data are in close agreement with our data (1.10 ± 0.37 mm/sec), despite the different methods used. In contrast, the mean velocity of leukocytes moving in the posterior choroid of the monkeys was 2.45 ± 0.48 mm/sec in the present study, approximately two times higher than the velocity in the retinal capillaries. The large choroidal vessels are at a depth different from that of the choriocapillaris. We observed leukocyte flow at a constant focal plane during a sequence of data collection. Thus, we may be biasing our observations to leukocytes that do not deviate significantly from the plane.
In the monkeys, the fluorescent leukocyte appeared abruptly on a video monitor, moved approximately 50 to 200 µm corresponding to the dimension of the lobules of the choriocapillaris,9 and disappeared abruptly. These movements seem to reflect the choroidal vasculature, in that feeding arteries and drainage veins are at a right angle to the choriocapillaris. Leukocytes in the choroidal arteries and veins move much faster than those in the capillaries. Indeed, we observed dim fluorescent dots moving too fast to be recognized in the still image, the quality of which was much lower than the real-time image. Fluorescence of leukocytes in the artery or vein of the deeper choroid may be more strongly blocked by much pigment of the choroid than that in the capillaries. Thus, we supposed that distinct fluorescent dots represent leukocytes passing through the choroidal capillaries. Japanese monkeys (Macaca fuscata) used in this study have much pigment in the choroid compared with humans. Chang et al.10 described that they used a higher dose of ICG (2 mg/kg) in the rhesus monkeys (Macaca mulatta) to gain high-contrast ICG angiographic images, compared with a dose of ICG (1 mg/kg) in human eyes. More distinct images of fluorescent leukocytes in the choroidal circulation might be gained in human eyes or other monkeys with less pigment. Further studies are necessary to prove the assumption that the distinct fluorescent spots seen in the present study are leukocytes passing through the choroidal capillary.
Transient plugging can be seen in the capillary under physiological and pathologic conditions.1 2 3 4 As described, transiently plugged leukocytes in the choriocapillaris were frequently seen in rabbits. The majority of them remained stagnant for less than 1 second. Plugging was sometimes seen in the monkeys. However, it is sometimes difficult to distinguish whether true plugging occurs or whether it is artifactual, due to movement of the leukocyte perpendicular to the choriocapillaris.
To our knowledge, this study is the first to visualize leukocytes moving in the choroidal microcirculation of pigmented animals including monkeys. ILA is available to evaluate leukocyte dynamics in vivo and noninvasively and would be useful for the experimental study of choroidal hemodynamics in pigmented subjects under pathologic conditions. Additionally, this method may be applicable in humans because of the minimal toxicity of ICG and holds a possibility of disclosing a role of leukocytes in the pathogenesis of various ocular diseases.
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Footnotes |
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Submitted for publication June 28, 1999; revised December 17, 1999 and April 14, 2000; accepted April 26, 2000.
Commercial relationships policy: N.
Corresponding author: Fumio Shiraga, Department of Ophthalmology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. fumio{at}cc.okayama-u.ac.jp
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This article has been cited by other articles:
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  M. Horiguchi, S. Nagata, N. Yamamoto, Y. Kojima, and Y. Shimada Kinetics of Indocyanine Green Dye After Intraocular Surgeries Using Indocyanine Green Staining Arch Ophthalmol, March 1, 2003; 121(3): 327 - 331. [Abstract] [Full Text] [PDF]
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