Effects of Granulocyte Colony Stimulating Factor on Retinal Leukocyte and Erythrocyte Flux in the Human Retina

Abstract

purpose. The blue-field entoptic technique was introduced more than 20 years ago to quantify perimacular white blood cell flux. However, a final confirmation that the perceived corpuscles represent leukocytes is still unavailable.

methods. The study design was randomized, placebo-controlled, and double masked with two parallel groups. Fifteen healthy male subjects received a single dose of granulocyte colony stimulating factor (G-CSF, 300 μg) and 15 other subjects received placebo. The following parameters were assessed at baseline and at 12 minutes and 8 hours after administration: retinal white blood cell flux, with the blue-field entoptic technique; retinal blood velocities, with bidirectional laser Doppler velocimetry; retinal venous diameter determined with a retinal vessel analyzer; and blood pressure and pulse rate determined by automated oscillometry and pulse oxymetry, respectively.

results. After 12 minutes, G-CSF reduced total leukocyte count from 5.5 ± 1.4 10⁹/L at baseline to 1.9 ± 0.4 10⁹/L. This was paralleled by a 35% ± 11% decrease in retinal white blood cell density. After 8 hours G-CSF increased total leukocyte counts to 20.0 ± 4.4 10⁹/L. Again, this increase in circulating leukocytes was reflected by an increase in retinal white blood cell density (110% ± 48%). All effects were significant at P < 0.001. By contrast, none of the other hemodynamic parameters was changed by administration of G-CSF.

conclusions. The results clearly indicate that the blue-field entoptic technique assesses leukocyte movement in the perimacular capillaries of the retina. Moreover, white blood cell density appears to adequately reflect the number of circulating leukocytes within the retinal microvasculature. Hence, an increase in retinal white blood cell density does not necessarily reflect retinal vasodilatation.

The blue-field entoptic phenomenon can be seen best by having the subject look into light with a narrow optical spectrum centered at a wavelength of 430 nm. Under such conditions, bright corpuscles are observed flying around the subject’s fovea. Most likely, this phenomenon is caused by the fact that red, but not white, blood cells absorb short-wavelength light. Thus, the passage of a white blood cell is perceived as a flying corpuscle. Riva and Petrig ¹ have developed a system that allows for the quantitative assessment of this flying-corpuscle phenomenon. However, a final proof that the perceived corpuscles represent leukocytes has not yet been provided. 

In the present study we used a new approach to pursue this hypothesis. We pharmacologically modulated the number of circulating leukocytes by infusion of granulocyte colony stimulating factor (G-CSF). G-CSF is an important regulator of the number and function of neutrophils. ^{2 3} It is used to treat neutropenia, but also for mobilization of stem cells or neutrophils. ⁴ The mechanisms by which G-CSF increases the number of circulating neutrophils is not well characterized. There is, however, some evidence that the early neutrophilia is caused by an influx of mature neutrophils from the bone marrow, ⁵ whereas the later phase is coupled to proliferation-dependent leukocytosis. ⁶  

In the present study, we investigated whether G-CSF infusion may alter the density and/or velocity of white blood cells, as assessed with the blue-field entoptic system. In addition, we measured retinal red blood cell velocities with laser Doppler velocimetry and retinal vessel diameters with a retinal vessel analyzer (Carl Zeiss; Oberkochen, Germany), to exclude that G-CSF has any effect on red blood cell flow in the retina. 

Methods

Subjects

The present study was performed in adherence to the tenets of the Declaration of Helsinki and the Good Clinical Practice guidelines. After approval of the study protocol by the Ethics Committee of the Vienna University School of Medicine and after written informed consent was obtained, 30 healthy male subjects were studied (age: 25 ± 4 years, mean ± SD). All subjects underwent a prestudy screening during the 4 weeks before the first study day, which included medical history and physical examination, 12-lead electrocardiogram, complete blood count, activated partial thromboplastin time, thrombin time, clinical chemistry (sodium, potassium, creatinine, uric acid, glucose, cholesterol, triglycerides, alanine aminotransferase, aspartate transcarbamylase, γ-glutamyltransferase, alkaline phosphatase, total bilirubin, and total protein levels), hepatitis A, B, and C and HIV serology, urinalysis, and an ophthalmic examination. Subjects were excluded if any abnormality was found during the pretreatment screening, unless the investigators considered an abnormality to be clinically irrelevant. In addition, subjects with ametropia of less than 3 D were included in the trial. During the last week after completion of the study, a follow-up safety investigation was scheduled, which included complete blood count, activated partial thromboplastin time, thrombin time, clinical chemistry as detailed earlier, and urinalysis. 

Study Design

Subjects were asked to refrain from alcohol and caffeine for at least 12 hours before trial days and were studied after an overnight fast. The study was performed in a randomized, placebo-controlled, double-masked, two-way parallel group design with a 1:1 randomization. On the morning of the study day, the participating subjects received either a single intravenous bolus infusion of G-CSF at a dose of 300 μg (Neupogen; Amgen-Europe BV, Breda, The Netherlands) or placebo (physiological saline solution). 

Description of Study Days

A resting period of at least 20 minutes in a sitting position was scheduled for all subjects. After stable hemodynamic conditions were achieved and ensured by repeated blood pressure measurements, baseline readings were obtained with the blue-field entoptic technique, the laser Doppler velocimeter, and the retinal vessel analyzer. Blood pressure, intraocular pressure, and pulse rate were also measured. Thereafter, G-CSF or placebo was administered, and hemodynamic parameters were measured after 12 minutes and 8 hours, respectively. A venous blood sample was also obtained at each time point to allow for a white blood cell count. 

Measurements

Blood Pressure and Pulse Rate.

Systolic, diastolic, and mean blood pressures were measured in the upper arm by an automated oscillometric device (HP-CMS monitor; Hewlett Packard, Palo Alto, CA). Pulse rate (PR) was automatically recorded from a finger pulse-oximetric device supplied with the monitor. 

Retinal Vessel Analyzer.

The retinal vessel analyzer (Carl Zeiss) is a commercially available system that comprises a fundus camera (FF 450; Carl Zeiss, Jena, Germany), a video camera, a real-time monitor, and a personal computer with analyzing software that provides accurate determination of retinal arterial and venous diameter. ⁷ Retinal vessel diameters are analyzed in real time with a maximum frequency of 50 Hz. For this purpose, the fundus is imaged onto the charge-coupled device chip of the video camera. The consecutive fundus images are digitized using a frame grabber. In addition, the fundus image can be inspected on the real-time monitor and, if necessary, stored on a video recorder. Evaluation of the retinal vessel diameters can be performed either on- or off-line from the recorded videotapes. Because of the absorbance properties of hemoglobin, each blood vessel has a specific transmittance profile. Measurement of retinal vessel diameters is based on adaptive algorithms using these specific profiles. Whenever a specific vessel profile is recognized, the retinal vessel analyzer is able to follow this vessel as long as it appears within the measurement window. If the requirements for the assessment of retinal vessel diameters are no longer fulfilled, as occurs during blinking, the system automatically stops the measurement of vessel diameter. As soon as an adequate fundus image is achieved again, measurement of vessel diameters restarts automatically. To select a region of interest, the user defines a rectangle on the screen of the real-time monitor. This window can include a retinal artery, a retinal vein, or both. As long as the vessels under study are within the selected rectangle, the system automatically corrects for eye movements. This is again permitted by the adaptive nature of the diameter analysis. Hence, vessel diameter can be recorded as a function of time, as well as a function of the position along the vessel. The system provides excellent reproducibility and sensitivity. ⁸ In the present study, major temporal veins were studied. Measurements of retinal venous diameters were taken between 1 and 2 disc diameters from the margin of the optic disc. Red blood cell velocity was measured at the same locations by using bidirectional laser Doppler velocimetry. 

Laser Doppler Velocimetry and Retinal Blood Flow.

The principle of blood flow velocity measurement by laser Doppler velocimetry (model 4000; Oculix Sarl, Arbaz, Switzerland) is based on the optical Doppler effect. Laser light, which is scattered by moving particles (e.g., erythrocytes) is shifted in frequency. This frequency shift is proportional to the blood flow velocity in the retinal vessel. The maximum Doppler shift corresponds to the centerline erythrocyte frequency. With bidirectional laser Doppler velocimetry, the absolute velocity in the retinal vessels can be obtained. ⁹ Retinal blood flow was calculated based on these measurements of maximum erythrocyte velocity (V _max) using laser Doppler velocimetry. Mean blood velocity in retinal veins was calculated as V _max/1.6. ^{10 11} Blood flow through a specific retinal vein i was then calculated as  

\[Q_{i}{=}(V_{\mathrm{max,}i}/1.6)\ {\cdot}\ ({\pi}\ {\cdot}\ d_{i}^{2}/4)\]

 

Blue-Field Entoptic Technique.

This noninvasive method is described in detail by Riva and Petrig. ¹² We used a commercially available system for the quantification of retinal white blood cell movement (Blue-field Simulator; Oculix Sarl). For determination of velocity and density of these flying corpuscles, a simulated particle field is shown to the subjects under study. By comparison with their own entoptic observation, subjects can adjust the number of white blood cells (WBCD) and the mean flow velocity (WBCV). In the present studies, at least five matching trials were performed by each subject. Only values with a coefficient of variation of less then 15% were considered accurate. Subjects who did not reach the required reproducibility were excluded from the study. 

Data Analysis

The effect of G-CSF on ocular hemodynamic parameters was calculated as the percentage of change from baseline readings. Statistically significant effects of G-CSF versus placebo were assessed with two-way ANOVA for repeated measures, by using the absolute values of all outcome parameters. Post hoc analyses were performed with unpaired t-tests. The Pearson product moment correlation was used to assess the association between total peripheral blood leukocyte count and WBCD, as measured with the blue-field entoptic technique. This was performed separately for the time points of measurement (12 minutes and 8 hours after G-CSF), but both groups (G-CSF and placebo) were pooled and analyzed together. Because two experimental groups were included in the same correlation analysis, we also applied a nonparametric test (Spearman correlation analysis). Data are the mean ± SD. P < 0.05 was considered the level of significance. 

Results

No adverse events were observed during the study, and leukocyte counts had returned to normal at the poststudy follow-up investigation. Baseline characteristics of the two study groups were comparable, showing no significant differences in ocular hemodynamic parameters (Fig. 1) , systemic hemodynamic parameters (Table 1) , or leukocyte count (Fig. 1) . The mean age (±SD) of the two study groups was also comparable (placebo and G-CSF groups: 25 ± 4 years). 

In the present trial, no consistent placebo effect was observed on any of the outcome parameters. Twelve minutes after G-CSF administration, a pronounced decrease in leukocyte count was observed (Fig. 1) . Total leukocyte count decreased from 5.5 ± 1.4 10⁹/L at baseline to 1.9 ± 0.4 10⁹/L after infusion of G-CSF. This effect was highly significant compared with that of the placebo (P < 0.001). The decrease in leukocyte counts was paralleled by a decrease in WBCD as assessed with the blue-field technique (−35% ± 11%; P < 0.001). Total leukocyte count (20.0 ± 4.4 10⁹/L) increased almost fourfold 8 hours after administration of G-CSF. This increase in total leukocyte count was again paralleled by an increase in WBCD (110% ± 48%). By contrast, retinal WBCV was not affected by administration of G-CSF. 

A significant association was observed between changes in total leukocyte count in venous blood and WBCD 12 minutes after G-CSF infusion (Fig. 2 , top; Pearson product moment: r = 0.73; P < 0.01; Spearman: r = 0.71; P < 0.01). The correlation line, however, was flatter than the 45° line, indicating that a twofold reduction in total leukocyte count led to only an approximate 50% reduction in WBCD, as perceived by the subjects under study. The same was true for the association between changes in total leukocyte count in venous blood and WBCD 8 hours after G-CSF infusion (Fig. 2 , bottom; Pearson product moment: r = 0.76; P < 0.01; Spearman: r = 0.74; P < 0.01). Compared with the 45° line the correlation line was again flattened, indicating that a change in leukocyte count is not fully reflected in WBCD. 

Administration of G-CSF did not exert any effect on retinal vessel diameter, retinal erythrocyte velocity as assessed with bidirectional laser Doppler velocimetry, or retinal blood flow (Fig. 1) . In addition, we did not note any effects of G-CSF on blood pressure or pulse rate (Table 1) , indicating an absence of direct systemic or retinal vascular effects. 

Discussion

The present trial strongly indicates that flying corpuscles as perceived when the subject looks into blue light, are intravascular leukocytes. Previous assumptions concerning the source of the blue-field entoptic phenomenon have been based on physiological and optical arguments. The particles are probably of vascular origin, because the pathway of the corpuscles shows similarities to otherwise invisible capillary loops, the corpuscles are not seen in an area around the fovea that could represent the foveal avascular zone, and the movement of the corpuscles is characterized by pulse-synchronous rhythmic velocity changes. ¹² Optical considerations regarding the source of the blue-field entoptic phenomenon were investigated in detail by Sullivan et al., ¹³ who used two microvascular preparations and video microscopy. The light source had a bandwidth comparable to that used in the present trial. Illumination of the isolated wing of the hibernating bat and the rat cremaster with light centered at a wavelength of 430 nm produced a field of moving particles. These particles appeared brighter than the plasma gaps between the red blood cells. Morphologic considerations indicated that the moving particles were leukocytes. A direct confirmation that the particles perceived with the subject looking into blue light represent leukocytes was not available, however, until the results of the present clinical trial. 

In the present study both the early reduction in leukocyte count and the late-phase increase in leukocyte counts were reflected in the measurements of WBCD. The observation that leukocyte count decreases early after administration of G-CSF is in keeping with several previous clinical trials. ^{5 14 15} The exact mechanism underlying this effect is unclear, but it has been hypothesized that the leukocytes are temporarily entrapped in the microvasculature, because of increased corpuscular stiffness. ¹⁶ However, correlation analysis of the present data revealed that a perfect correlation was not achieved, nor did the correlation line reach an inclination of 45°. Apparently, the variability in both leukocyte count determinations from venous blood samples and WBCD measurements with the blue-field technique reduced the correlation coefficient. 

There may, however, be other method-related factors that hamper perfect correlation. Obviously, the blue-field entoptic technique more adequately represents leukocyte movement within the microvasculature. Blood drawn from an antecubital vein does not necessarily reflect the total number of leukocytes, because of phenomena such as leukocyte margination, particularly in the microvessels. Moreover, the different sizes of neutrophils, lymphocytes, and granulocytes may result in different flowing and rolling properties in the retina and the forearm. This effect may be enhanced by differences in adhesion molecule expression in different tissues. In contrast, it is noteworthy that WBCD underestimated the effect at times of leukopenia and leukocytosis. This may reflect that the more circulating leukocytes perceived by the subject, the less leukocytes are circulating in total. In other words the large number of leukocytes circulating 8 hours after G-CSF infusion may, per se, reduce the fraction of perceived leukocytes when the subject looks into blue light. 

Nevertheless, the results of the present study indicate that WBCD is at least a semiquantitative measure of the number of circulating leukocytes within the retinal microvasculature. Hence, it is feasible to calculate white blood cell flux as the product of WBCV and WBCD, as has been the practice previously. ^{17 18 19 20} Care has to be taken, however, to use white blood cell flux as assessed with the blue-field technique as a measure of retinal blood flow. The present study indicates that an increase in WBCD may arise either from retinal vasodilation or from leukocyte cell recruitment. This must be taken into account in pharmacodynamic studies, in which the administered drug may alter the total number of leukocytes, and in cross-sectional studies, in which differences in WBCD may simply reflect differences in total circulating leukocytes between groups. In addition, it has previously been shown that the velocity of leukocytes in retinal capillaries is slower than that of erythrocytes. ²¹  

In conclusion, in the present trial G-CSF infusion in human subjects altered WBCD, as assessed with the blue-field entoptic phenomenon, whereas WBCV was unaffected. Neither systemic hemodynamic parameters nor retinal red blood cell flux was affected by infusion of G-CSF. These data clearly indicate that leukocyte movement underlies the blue-field entoptic phenomenon. In addition, our data indicate that an increase in retinal WBCD did not necessarily reflect retinal vasodilatation. Combination of the blue-field technique and optical Doppler technology may provide a unique opportunity to study the interaction between erythrocytes and leukocytes in the human retina. 

 Submitted for publication November 1, 2001; revised January 7, 2002; accepted January 15, 2002.

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Figure 1.

 

Time course of leukocyte counts, WBCV, WBCD, mean retinal red blood cell velocity (Vel), retinal venous diameter (Diameter), and retinal blood flow through the selected vein (Flow) after administration of G-CSF (▴) or placebo (▿). Data are the mean ± SD of 15 readings in each group. *Significant effects of G-CSF versus placebo.

Table 1.

 

Systemic Hemodynamic Variables after Administration of G-CSF or Placebo

	Baseline	12 Minutes	8 Hours
G-CSF Group (n = 15)
Systolic blood pressure (mm Hg)	118 ± 10	120 ± 9	119 ± 9
Diastolic blood pressure (mm Hg)	64 ± 9	63 ± 9	63 ± 9
Pulse rate (beats/min)	70 ± 11	67 ± 13	77 ± 11
Placebo group (n = 15)
Systolic blood pressure (mm Hg)	114 ± 10	117 ± 12	118 ± 10
Diastolic blood pressure (mm Hg)	62 ± 9	67 ± 8	64 ± 11
Pulse rate (beats/min)	64 ± 11	67 ± 10	72 ± 12

 Data are the mean ± SD of readings in 15 subjects at the time points shown.

Figure 2.

 

Association between the change in leukocyte count and the change in WBCD. The correlations were independently performed for both time points (top, 12 minutes; bottom, 8 hours: lower panel), but both study groups (placebo and G-CSF) were pooled. The correlation line and the 95% confidence intervals are presented.