Abstract
Purpose.:
The present study tested the hypothesis that human choroidal blood flow (ChBF) regulation in the face of changes in ocular perfusion pressure (OPP) may be modified by a drug-induced decrease in intraocular pressure (IOP).
Methods.:
This hypothesis was tested in a double-masked, randomized, placebo-controlled, parallel-group trial in 24 healthy volunteers. OPP was manipulated by 6 minutes of squatting and a subsequent period of artificial increase in IOP induced with a suction cup. These interventions were repeated after 14 days of treatment with either latanoprost or placebo. ChBF was measured continuously with a portable laser Doppler flowmeter.
Results.:
As expected, latanoprost significantly reduced IOP compared with placebo (P = 0.008). The relative increases in OPP during squatting (P = 0.97) and an artificial IOP increase (P = 0.75), however, were comparable after placebo and latanoprost. The response of ChBF was, in contrast, different between the two treatment groups. During the squatting-induced elevation of OPP, ChBF increased less after latanoprost than after placebo treatment (P = 0.049). During the suction cup–induced increase in IOP, the decrease in ChBF was less pronounced after latanoprost than after placebo (P = 0.026). Latanoprost, however, did not modify baseline ChBF at rest (P = 0.30).
Conclusions.:
The data indicate that latanoprost improves ChBF regulation during both an increase and a decrease in OPP. Since latanoprost did not affect baseline ChBF, the authors assume that this effect is related to the decrease in IOP. This finding has important implications for understanding the relation between IOP and vascular factors in glaucoma, because it indicates that a reduction in IOP itself improves ChBF regulation. (ClinicalTrials.gov number, NCT00712400.)
There has been a long-standing discussion on the regulatory capacity of the choroidal circulation.
1 –18 We have recently shown that the regulatory capacity of the human choroid in face of changes in ocular perfusion pressure (OPP) may differ if either arterial blood pressure or intraocular pressure (IOP) is modified.
17 More specifically, we have shown that, during a combined-exercise–induced increase in mean arterial pressure (MAP) and a suction cup–induced increase in IOP, choroidal blood flow (ChBF) is regulated better during the former than during the latter. We have speculated that this behavior is related to either sympathetic vasoconstriction in response to isometric exercise on the arterial side of the vascular system or to a myogenic response.
17
In a clinical trial in patients with primary open-angle glaucoma or ocular hypertension, the association between OPP and ocular blood flow parameters was compared before and after pressure reduction with either dorzolamide or timolol.
19 –21 With both drugs, the correlation line between OPP and ocular blood flow parameters became less steep, indicating better blood flow regulation. Since only dorzolamide, not timolol, increased blood flow at baseline, we concluded that the change in the pressure-flow characteristics was due to the ocular hypotensive effects of the drug.
In the present study we wanted to get further insight into ChBF regulation and the complex interaction between OPP, MAP, IOP, and choroidal perfusion. We hypothesized that a reduction in IOP may alter ChBF regulation in the face of changes in OPP. To test this hypothesis, we performed a randomized, double-masked, parallel-group study in healthy subjects. The participants received either latanoprost, a prostaglandin analogue, to lower IOP, or a placebo for 14 days, and the regulation of ChBF was tested before and after this period. The regulation during an increase in OPP was studied when MAP was increased by isometric exercise. The regulation during a decrease in OPP was studied when IOP was increased with a suction cup.
The present study was performed in compliance with the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines. The study protocol was approved by the Ethics Committee of the Medical University of Vienna. A total of 24 healthy male subjects aged between 19 and 35 years participated. The nature of the study was explained to all subjects, and they gave written consent to participate. Each subject passed a screening examination including medical history and physical examination; 12-lead electrocardiogram; complete blood count; activated partial thromboplastin time; thrombin time; fibrinogen; clinical chemistry (sodium, potassium, creatinine, uric acid, glucose, cholesterol, triglycerides, alanine amino-transferase, aspartate transcarbamylase, γ-glutamyltransferase, alkaline phosphatase, total bilirubin, and total protein); hepatitis A, B, and C and HIV serology; urine analysis; and a urine drug-screening. Subjects were excluded if any abnormality was found as part of the pretreatment screening, unless the investigators considered the abnormality to be clinically irrelevant. Moreover, an ophthalmic examination, including slit lamp biomicroscopy and indirect funduscopy, was performed. Inclusion criteria were normal ophthalmic findings, ametropia of less than 3 D, and anisometropia of less than 1 D.
The study was performed in a randomized, double-masked, placebo-controlled design in two parallel groups. One group received latanoprost (Xalatan 0.005%; Pfizer, New York, NY) once daily for 14 days. The other group received a placebo (physiologic saline solution), which was identical in appearance. All subjects were asked to refrain from alcohol and caffeine for at least 12 hours before trial day. In addition, the subjects were instructed to have 7 to 8 hours of sleep and a light breakfast before arriving at the Department of Clinical Pharmacology on the trial day. On the first study day (day 1), after a resting period of at least 20 minutes in a sitting position, the participants underwent baseline measurements of ocular and systemic hemodynamics and IOP. Thereafter, the isometric exercise stimulation period was started. First, a 3-minute continuous baseline recording of ChBF was scheduled. After this baseline recording, each subject engaged in 6 minutes of isometric exercise. This period of isometric exercise consisted of squatting in a position where the upper and the lower legs formed approximately a right angle, to increase mean arterial blood pressure. During this squatting period, continuous measurement of ChBF was performed. At the end of the squatting period, IOP was measured again. Blood pressure and pulse rate were assessed every minute throughout these experiments.
Thereafter, another resting period of 30 minutes was scheduled to allow for normalization of blood pressure and pulse rates. Afterward, ChBF was measured continuously for three minutes at baseline. Then, the suction cup was applied using a suction force of 50 mm Hg. The suction force was then increased in three consecutive steps to 75, 100, and 125 mm Hg. Each suction level was maintained for 2 minutes during which ChBF was continuously measured. After another resting period, a final suction cup period was scheduled that followed exactly the time course of the first suction cup period. During this period, IOP was measured at each suction cup level by applanation tonometry.
After 14 days of eye drop instillation with either latanoprost or placebo a second study day was scheduled for each subject. The time course of this study day (day 2) was identical with that for study day 1. All IOP measurements were performed by investigators who were not involved in any other parts of the study including ChBF data collection, to allow for double-masked conditions.
Systemic Hemodynamics.
Brachial artery blood pressures: systolic blood pressure (SBP), diastolic blood pressure (DBP), and MAP were monitored on the upper arm by an automated oscillometric device. The pulse rate (PR) was automatically recorded from a finger pulse oximeter (HP-CMS; Hewlett Packard, Palo Alto, CA).
Laser Doppler Flowmetry.
Continuous measurements of subfoveal ChBF were performed by laser Doppler flowmetry (LDF), as described in principle previously.
22 With this technique, the vascularized tissue is illuminated by coherent laser light. Light scattered by the moving red blood cells undergoes a frequency shift. In contrast, static tissue scatterers do not change the light frequency, but lead to randomization of light directions impinging on red blood cells. Hence, red blood cells receive light from numerous random directions. As the frequency shift is dependent, not only on the velocity of the moving red blood cells, but also on the angle between the incident and the scattered light, scattering of the light in tissue broadens the Doppler shift power spectrum. From this spectrum, hemodynamic parameters can be determined based on a theory of light scattering in tissue. In the present study, a compact laser Doppler flowmeter, which has been described in detail previously, was used.
23,24 All measurements were performed in the fovea by asking the subject to directly fixate at the beam. The fovea was chosen, because the retina is avascular in this region. The following blood flow parameters were obtained: ChBF, velocity (VEL), and volume (VOL). VEL is the mean velocity of the red blood cells moving in the sampled tissue proportional to the mean Doppler frequency shift. VOL is the number of moving red blood cells in the sampled tissue. ChBF was calculated as the product of VEL and VOL.
Intraocular Pressure.
A slit lamp–mounted Goldmann applanation tonometer was used to measure IOP. Before each measurement, one drop of 0.4% benoxinate hydrochloride combined with 0.25% sodium fluorescein was used for local anesthesia of the cornea.
Suction Cup Method.
The IOP was raised applying a method described by Ulrich and Ulrich.
25 In the present study, we used an automatic suction pump that is connected by plastic tubing to a rigid plastic suction cup. After topically applied local anesthesia, a standardized 11-mm diameter suction cup was placed on the temporal sclera with the anterior edge at least 1 mm from the limbus. The vacuum was increased in increments from 50 to 125 mm Hg.
OPP was calculated as ⅔MAP − IOP.
26 All blood flow values measured at OPP below 10 mm Hg were excluded from statistical analysis because of problems in LDF signal analysis at very low flow rates, as discussed in detail by Riva et al.
27
A repeated-measures ANOVA model was used to analyze data. Data during the IOP increase and the squatting periods were analyzed separately. For both analyses the pretreatment and posttreatment data were included in one model. Accordingly the repeated-measures ANOVA model during IOP increase included 10 values for each dependent variable, and the model during squatting included 14 values for each dependent variable. Differences between latanoprost and placebo were calculated based on the interaction between time and treatment. Post hoc analyses were performed using planned comparisons. For this purpose, the time effect was used to characterize the effect of IOP increase and squatting on the outcome parameters. The treatment effect was used to characterize the effect of latanoprost on resting values. For data description, the percentage of change over baseline was calculated. P < 0.05 was considered the level of significance (CSS Statistica for Windows; StatSoft Inc., Tulsa, OK).
The baseline characteristics of the subjects are presented in
Table 1. There were no significant differences between the group of subjects receiving placebo and the group receiving latanoprost. All participating subjects finished the study as scheduled, and no adverse reactions were noted except mild localized conjunctival hyperemia after suction cup application.
The effect of latanoprost and placebo on all outcome variables is shown in
Table 2. Neither of the two treatments affected blood pressure or PR. As expected, latanoprost caused a decline in IOP that was significant versus placebo (
P = 0.013). On average, IOP was reduced by 17.9% (−2.5 ± 2.8 mm Hg), resulting in an average increase in OPP of 6.3% (+4.2 ± 5.9 mm Hg;
P = 0.004 versus placebo). ChBF, however, was unchanged at baseline.
The effect of squatting on OPP and ChBF is shown in
Figure 1. During all squatting periods, there was a significant increase in OPP (each
P < 0.001 versus baseline) and ChBF (each
P < 0.001 versus baseline). The increase in ChBF was less pronounced in both groups than the increase in OPP, indicating some degree of blood flow regulation.
The response of OPP to squatting was not significantly different between the pretreatment periods and the posttreatment periods with either latanoprost or placebo (P = 0.97). Posttreatment mean OPP (SD) increased on average by 46.5% (±17.5%) in the placebo group and 51.2% (±19.8%) in the latanoprost group compared with baseline. By contrast, there was a significant difference between the response of ChBF to squatting after latanoprost treatment and that after placebo treatment. Whereas the response after placebo was not different compared with that in the pretreatment period, the increase in mean ChBF after latanoprost treatment was less pronounced (10.9% ± 5.9%; P = 0.049 between groups) compared to both the pretreatment (19.8% ± 9.9%) and placebo (19.0% ± 7.5%) responses.
The effect of the suction cup application on IOP, OPP, and ChBF is show in
Figure 2. Although subjects receiving latanoprost started at lower IOPs, the increase during suction cup application was parallel. As expected, this result was associated with a decrease in OPP and ChBF (each
P < 0.001 versus baseline). The relative change in ChBF was less pronounced than the decrease in OPP (73.7% ± 12.3%), indicating some regulatory capacity of the choroid under these circumstances. The response of OPP was not different between the pre- and posttreatment periods (
P = 0.75). The response of ChBF to the artificial increase in IOP was not altered during placebo administration. By contrast, the reduction in ChBF after administration of latanoprost was significantly less pronounced (26.8% ± 15.3%) compared with the pretreatment response (40.0% ± 11.5%) and the placebo response (41.0% ± 12.4%;
P = 0.024 between groups).
In keeping with results from previous studies performed by our group as well as other investigators, ChBF showed some regulatory capacity in response to OPP changes induced by isometric exercise
10,12 –15 or an artificial IOP increase.
8,17 In the present study we hypothesized that the counterregulatory capacity of the choroidal vasculature may be altered by a lower IOP after a 14-day treatment period with latanoprost. The response of ChBF to both interventions was significantly different with the prostaglandin analogue compared with placebo, although the relative change in OPP was comparable. During isometric exercise, the increase in ChBF was less pronounced after latanoprost than after placebo administration. This result indicates that the increase in vascular resistance was more pronounced after latanoprost due to more distinctive vasoconstriction in the face of the OPP increase. During the artificial increase in IOP, the decrease in ChBF was less pronounced after latanoprost than after placebo. Considering that this decrease was observed at a comparable OPP decrease, it indicates more counterregulatory vasodilatation after a latanoprost-induced decrease in IOP compared with placebo.
How can these results be interpreted? After topical administration of antiglaucoma drugs, two distinct mechanisms have to be distinguished.
28,29 On the one hand, the drug itself may induce direct vasomotor effects if it reaches the posterior pole of the eye at a sufficient concentration. In the present study we deem it unlikely that this is the case, because latanoprost did not affect baseline ChBF. Hence, the different behavior of the choroidal vasculature in face of the changes in OPP after latanoprost is unlikely to be related to a direct vasomotor effect, because the drug did not alter baseline vascular tone. On the other hand, any topical glaucoma drug alters OPP, because of the ocular hypotensive action. Since choroidal venous pressure is very close to IOP over a wide range of pressures,
30 any decrease in IOP directly transfers into an increase in OPP. The observation that ChBF reacts differently to changes in OPP at comparable ChBF levels at baseline may well be related to the increase of OPP induced by latanoprost.
We have shown in two studies that ChBF may be regulated better at lower levels of IOP than at higher levels of IOP. In a study in healthy subjects, we investigated the effects of combined increase in IOP using a suction cup and an increase in MAP with isometric exercise. In that study, the regulatory mechanisms of choroidal circulation compensated better for an increase in blood pressure than for an increase in IOP.
17,31 The mechanism that may be responsible for this behavior was first formulated for the choroid by Kiel
4 –7 based on his results in the rabbit. This method assumes that the choroid shows at least to some degree a myogenic response. Hence, changes in transmural pressure are responsible for smooth muscle constriction in response to an OPP increase due to the IOP-lowering effect of latanoprost. Consequently, the system is at a stage of relative vasoconstriction after latanoprost, compared with placebo or baseline conditions. When IOP is then artificially increased by use of a suction cup, the vasodilator reserve will be higher after latanoprost than under placebo, resulting in better counterregulation, as observed in the present study. In case of the isometric exercise period, however, the arteriovenous pressure difference is larger after latanoprost than after placebo. In this case, a more pronounced myogenic response will be initiated because of the increase in transmural pressure in the arterial system supplying the choroid resulting in vasoconstriction and a better regulatory capacity.
Several limitations need to be considered when discussing the results of our experiments. Choroidal vessels show a rich innervation,
32 –35 and stimulation of the sympathetic system induced pronounced vasoconstriction.
36 –38 Obviously, the neural input into the system cannot be eliminated in a human experiment, and it is likely that this influenced the response of the choroidal vasculature in our studies. As such, one should not refer to the regulatory behavior of the choroid in the present study as autoregulation. The fact that latanoprost altered the regulatory behavior in response to both an increase and a decrease in OPP indicates, however, that the mechanism underlying blood flow regulation in the present study is not entirely neural.
In addition, we cannot entirely exclude that small amounts of the prostaglandin analogue reached the posterior pole of the eye, influencing vascular tone to a small degree. In this context, the sample size of the present study allowed only for detection of changes in ChBF at baseline in the order of 12%.
39 Previous studies on the effect of latanoprost on ocular blood flow are equivocal.
40 –49 Most studies that indicate increased blood flow after latanoprost treatment, however, were based on pneumotonometric studies, in which the effect on the IOP on the ocular pressure pulse was not adequately taken into account.
50,51 On the basis of the results of our studies with β-receptor antagonists and carbonic anhydrase inhibitors,
21 we deem it unlikely, however, that a direct vasomotor effect contributes to the altered blood flow response in the face of the OPP changes. On the other hand, one needs to consider that the effects of latanoprost on IOP (−2.5 mm Hg) and OPP (+4.2 mm Hg) were small. It is striking that such small changes in IOP can cause pronounced changes in ChBF regulation. Further studies with alternative methods to modify IOP and OPP are needed, to gain more insight into this topic.
Finally, assessment of OPP may have limitations, particularly in connection with the suction cup technique. OPP is calculated as ⅔MAP − IOP, but this equation may not be appropriate at high IOP levels because of the compression of larger vessels entering the sclera. There is, however, no equation that accounts for this problem. The limitation applies to both the latanoprost and the placebo groups and therefore does not affect the primary hypothesis of the study.
Our results may have implications for the understanding of current glaucoma therapy. It has been suggested that vascular factors play a role in the pathogenesis of the disease, particularly vascular dysregulation and low OPP.
52,53 Part of the vascular dysregulation may result from systemic disease and endothelial dysfunction.
54 –57 There is, however, accumulating evidence from our previous studies and the present study that the level of IOP is a major determinant of the regulatory capacity of the choroid. Whether this concept is also true for optic nerve head blood flow is currently under investigation in our laboratory. Most studies focused on the effect of antiglaucoma drugs on baseline ocular blood flow, and only one study is available indicating that an increase in blood flow is indeed beneficial in terms of visual field preservation.
58 Our data indicate that future studies should particularly focus on blood flow regulation and the relation to progression of glaucoma. Moreover, the present results suggest that any reduction in IOP is by itself associated with an improvement in ocular blood flow regulation.
In conclusion, the present study indicates that administration of latanoprost improves ChBF regulation during both an increase and a decrease in OPP. Since latanoprost did not affect baseline blood flow we assume that the ocular hypotensive effect of latanoprost is responsible for this improvement in regulation compatible with a myogenic mechanism. These results may improve our understanding of ocular blood flow regulation and glaucoma, because any reduction in IOP, proven to be beneficial for slowing down the progression of the disease, appears to be associated with improved ChBF regulation.
Supported by the Austrian National Bank, project number 11309.