- Email: [email protected]
Color Doppler Ultrasound Analysis of Ocular Circulation After Topical Calcium Channel Blocker
Color Doppler Ultrasound Analysis of Ocular Circulation After Topical Calcium Channel Blocker PETER A. NETLAND, MD., CYNTHIA L. GROSSKREUTZ, MD., GILBERT T. FEKE, PH.D., AND LOIS J. HART
• PURPOSE: To investigate the effect of topical administration of the calcium channel blocker verapamil on intraocular pressure and retrobulbar hemodynamics. • METHODS: In this randomized, prospective, double-masked study, we examined the effects of single-dose topical administration of verapamil in ten normal human volunteers by using color Doppler ultrasound imaging to measure hemodynamic parameters. Limitations of this study in clude single-dose application of verapamil and relatively small sample size. • RESULTS: N o systemic effect on heart rate or blood pressure was detected after administration of topical verapamil. The intraocular pressure signif icantly decreased compared with baseline two hours after topical 0.125% and 0.25% verapamil (P = .015 and . 0 4 0 , respectively). Pourcelot's ratio, an index of vascular resistance, measured in the central retinal artery was significantly reduced after topical application of 0.125% verapamil (P = .008). The change in Pourcelot's ratio primarily resulted from an increased end diastolic velocity in the central retinal artery. N o significant differenc es compared with baseline values were detected in the color Doppler ultrasound measurements of the Accepted for publication Dec. 27, 1994. From the Department of Ophthalmology, Massachusetts Eye and Ear Infirmary (Drs. Netland and Grosskreutz and Ms. Hart), and Schepens Eye Research Institute (Dr. Feke), Harvard Medical School, Boston, Massachusetts. This study was supported by the Massachusetts Lions Eye Research Fund, Northboro, Massachusetts, and CooperVision Pharmaceuticals, San Clémente, California. Reprint requests to Peter A. Netland, M.D., Department of Oph thalmology, Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 02114; fax: (617) 573-3707.
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posterior ciliary arteries and the central retinal vein two hours after topically administered verapamil. • CONCLUSIONS: Topical administration of ver apamil decreases intraocular pressure and alters ocular hemodynamics, reducing the vascular re sistance index in the central retinal artery.
C
ALCIUM CHANNEL BLOCKERS ARE USED WIDELY for the treatment of cardiovascular disorders, including angina pectoris, cardiac arrhyth mias, systemic hypertension, and Raynaud's phenom enon. 1 These drugs reduce vascular resistance and help prevent vasospasm. The ocular effects of calcium channel blockers are of interest for their potential clinical role, in particular for the treatment of glauco ma patients. Systemic administration of calcium channel block ers in humans has generally resulted in a decreased intraocular pressure.2'4 In ocular hypertensive pa tients, topical verapamil causes a marked and sus tained reduction of intraocular pressure.5,6 This reduc tion of intraocular pressure suggests that verapamil might be used to treat hypertensive glaucoma. In addition to an effect on intraocular pressure, calcium channel blockers may have other beneficial effects in glaucoma because they inhibit vasospasm or enhance optic nerve blood flow, which may be particularly influential in low-tension glaucoma. In a previous retrospective cohort study, we found that the use of calcium channel blockers is associated with a slowed progression of low-tension glaucoma.7 Sustained improvement of visual fields has been described after administration of calcium channel
© AMERICAN JOURNAL OF OPHTHALMOLOGY ii9:694-7oo
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blockers to patients with low-tension glaucoma.8 In an experimental study in cats, intravenous nicardipine caused an increased optic nerve head blood flow as measured by laser Doppler flowmetry.9 Color Doppler ultrasound imaging allows real-time B-scan ultrasound imaging of anatomic structures in the orbit with superimposed color-coded Doppler frequency shifts resulting from blood flow, allowing localization of vessels and measurement of vascular blood velocities.10'12 Using this noninvasive tech nique, we studied the effects of the topical calcium channel blocker verapamil on ocular circulation. We performed a prospective, double-masked, randomized study in normal human volunteers of the effect of single-dose topical verapamil on systemic blood pres sure and heart rate, intraocular pressure, and color Doppler ultrasound measurements of ocular blood vessels.
SUBJECTS AND METHODS VERAPAMIL OPHTHALMIC FORMULATION, PROVIDED BY
CooperVision Pharmaceuticals (San Clémente, Cali fornia), contained 0.003% benzalkonium chloride and 0.063%, 0.125%, or 0.25% verapamil. The placebo was identical to the vehicle of the verapamilcontaining solution. The drug and placebo drops were provided with coded labels so that neither the investi gators nor the patient had any knowledge of which eye was receiving the drug or which strength of drug. Ten volunteers in good health (seven women and three men with a mean ± S.D. age of 29 ± 6 years), who were not receiving any ocular or systemic medi cations, were recruited for the study. After informed consent was granted, baseline intraocular pressure, heart rate, systemic blood pressure, and color Doppler ultrasound values were measured. Eyedrops contain ing 0.063%, 0.125%, or 0.25% verapamil were in stilled randomly and in a double-masked fashion in one eye, and placebo was administered to the fellow eye. Intraocular pressure, heart rate, systemic blood pressure, and color Doppler ultrasound measurements were repeated two hours after instillation of eyedrops containing verapamil or placebo. Each subject was studied on three days, separated by at least one week between measurements, to obtain ten measurements for each of the three doses of verapamil. The order of VOL.119, No. 6
the three doses was randomized on the three days, with the examiners masked as to which doses were administered to each subject. Baseline measurements were repeated on each visit. Color Doppler ultrasound imaging was performed with a Siemens Quantum 2000 (Issaquah, Washing ton) using a 7.5-MHz transducer. The flow setting was for slow flow, and the sample volume was 1.2 mm3. The scan plane was horizontal through the optic nerve. The probe was carefully placed over the upper eyelid with minimal pressure. All color Doppler ultrasound measurements were performed by one of us (L.J.H.) without knowledge of which eyes had been treated with verapamil. Measurements of the peak systolic velocity, end diastolic velocity, and Pourcelot's ratio were performed in duplicate for the central retinal artery, nasal and temporal posterior ciliary arteries, and central retinal vein. Pourcelot's ratio was calculated as (peak systolic velocity minus end diastolic flow velocity) divided by peak systolic velocity.13 Pourcelot's ratio, which reflects vascular resistance, tends to be independent of the Doppler angle. The values of Pourcelot's ratio can vary from 0 to 1, with higher numbers indicating greater vascular resistance. The reproducibility of color Doppler ultrasound has been studied, with coefficients of variation in the ophthalmic artery ranging from 4% for the resistive index to 11% for peak systolic velocity.1415 Central retinal artery and vein measurements were performed within the less echogenic optic nerve shadow, ap proximately 2 mm posterior to the globe. Posterior ciliary vessels were located either temporally or nasal ly adjacent to the optic nerve shadow. We attempted to study the same site for subsequent measurements in each subject. The spectral measurement was anglecorrected to the longest axis obtainable, and Doppler angle did not vary on repeated measurements in the same subjects. The two-tailed paired t-test was used to evaluate the statistical significance of the change from baseline of each of the measured quantities and the differences in the changes between eyes receiving verapamil and eyes receiving placebo. In the central retinal artery measurements, the power of the test to determine a 12% change of Pourcelot's ratio was .57. Spearman correlation analysis was used to evaluate the relation ships between the changes in intraocular pressure and
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695
TABLE 1 SYSTEMIC HEART RATE AND BLOOD PRESSURE AFTER APPLICATION OF TOPICAL VERAPAMIL 0.063% VERAPAMIL
Heart rate (beats/min)* Systolic blood pressure (mm Hg)· Diastolic blood pressure (mm Hg)*
BASELINE
AFTER APPLICATION
72 ± 4 114 ± 2 69 ± 2
ne + 2
0.125% VERAPAMIL
0.25° » VERAPAMIL
BASELINE
AFTER APPLICATION
BASELINE
AFTER APPLICATION
68 ± 3 115i2 70 ± 2
70 ± 3 118 ± 2 72 ± 3
69 ± 3 115 ± 3 73 ± 3
68 ± 4 114 ± 2 73 ± 2
70 ± 4 71 ± 2
'Values shown are mean ± S.E. There was no statistically significant difference after topical application of verapamil.
the changes in the hemodynamic parameters. P < .05 were considered significant.
RESULTS THE
EFFECTS OF
TOPICALLY ADMINISTERED
0.063%,
0.125%, and 0.25% verapamil on systemic heart rate and blood pressure were studied (Table 1). Compared with baseline values, no statistically significant differ ences in heart rate were noted two hours after any of these doses of topical verapamil. Similarly, systolic and diastolic blood pressure measurements did not vary significantly two hours after application of topi cal verapamil. The mean baseline intraocular pressure was 13.9 ± 0.7 mm Hg (mean ± S.E.) for all placebo- and verapamil-treated eyes. As shown in Figure 1, the mean intraocular pressure was reduced compared with baseline in eyes treated with topical verapamil. This reduction of intraocular pressure was statistically significant after administration of 0.125% (P = .015) and 0.25% (P = .040) verapamil, but not after administration of 0.063% verapamil. The intraocular pressure of the fellow eyes, which were treated with placebo, did not vary significantly compared with baseline. Pourcelot's ratio, an index of vascular resistance, was measured two hours after topical administration of 0.063%, 0.125%, and 0.25% verapamil (Fig. 2). As shown in Table 2, the mean Pourcelot's ratio of the central retinal artery was significantly reduced com pared with baseline after administration of 0.125% verapamil (P = .008). At the higher 0.25% dose, Pourcelot's ratio was also decreased compared with baseline (Fig. 2), but this difference was not statisti 696
cally significant. There was no significant change in Pourcelot's ratio in the fellow placebo-treated eye at any of the examinations. On average, as shown in Table 2, systolic and diastolic velocities in the central retinal artery in creased after topical verapamil. As shown in Figure 3, the percent increase of the diastolic velocities was greater than that of the systolic velocities. The mean ± S.E. of the percent increase in diastolic velocity in the central retinal artery was 56% ± 25% after administration of 0.125% verapamil (P = .12) and
■ verapamil D placebo
o
0.063%
0.125%
0.25%
Verapamil Concentration
Fig. 1 (Netland and associates). Effect of topical verapa mil on intraocular pressure. The percent change of intraocular pressure (mean ± S.E.) from baseline is shown two hours after three different doses of verapamil for the treated eye and the fellow eye that received a placebo. The reduction of intraocular pressure was significant compared with baseline for the 0.125% and 0.25% doses of verapamil (P = .015 and .040, respec tively).
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baseline 2 hours
S. 0.70
0.063%
0.125%
0.25%
Verapamil Concentration
Fig. 2 (Netland and associates). Color Doppler ultra sound measurement of Pourcelot's ratio of the central retinal artery at baseline and two hours after topical administration of 0.063%, 0.125%, and 0.25% verapa mil (mean ± S.E.). The Pourcelot's ratio, a measure of vascular resistance, was significantly decreased com pared with baseline after 0.125% verapamil (P = .008).
35% ± 24% after administration of 0.25% verapamil (P = .16). In the eye that received placebo, the systolic velocity of the central retinal artery was increased by 15% ± 6% (P = .030) after 0.25% verapamil in the fellow eye, suggesting a possible crossover effect. Spearman correlation analysis showed no statistically significant relationships be tween the changes in intraocular pressure and the changes in systolic velocity, diastolic velocity, or Pourcelot's ratio in either eye at any of the concentra tions of verapamil. Color Doppler measurements were also performed in the temporal and nasal posterior ciliary arteries and in the central retinal vein. In these vessels, no significant differences compared with baseline meas urements were found in the mean peak systolic velocity, end diastolic velocity, or Pourcelot's ratio two hours after topical administration of 0.063%, 0.125%, or 0.25% verapamil. Color Doppler ultra sound measurements after administration of 0.125% verapamil are shown in Table 3.
DISCUSSION CALCIUM CHANNEL BLOCKERS SLOW THE PROGRESSION
effects on the visual fields of glaucoma patients.7,8 Possible mechanisms that may explain these effects include prevention of vasospasm or enhancement of ocular blood flow. Color Doppler ultrasound imaging is a noninvasive technique that may be used to assess the retrobulbar circulation.10'12 In this study, we exam ined the ocular and systemic effects of topically applied verapamil in a prospective, double-masked, randomized study in normal human volunteers. We found no effect on systemic blood pressure or heart rate but did find decreased intraocular pressure after administration of topical verapamil. Using color Doppler ultrasound imaging, we also demonstrated changes in ocular hemodynamics, with a reduction of the resistive index in the central retinal artery. Although systemic calcium channel blockers are known to decrease vascular resistance and reduce blood pressure, we found no change in systemic blood pressure or heart rate after topical administration of verapamil. Abelson, Gilbert, and Smith5 also found no change in blood pressure or heart rate after a single drop of topically applied verapamil. Goyal and associ ates,6 however, found decreased heart rate and blood pressure after two weeks of treatment with topical verapamil. This difference may be explained by the longer period of treatment or the higher baseline heart rates and blood pressures in the study by Goyal and associates. There may also have been differences TABLE 2 COLOR DOPPLER ULTRASOUND MEASUREMENTS IN THE CENTRAL RETINAL ARTERY TWO HOURS AFTER APPLICATION OF TOPICAL 0.125% VERAPAMIL
Peak systolic velocity (cm/sec)* End diastolic velocity (cm/sec)* Pourcelot's ratiof
BASELINE
AFTER APPLICATION OF VERAPAMIL
8.79 ± 0.44
8.87 ± 0.60
2.17 ± 0.34
2.78 ± 0.27
0.76 ± 0.03
0.67 ± 0.03
"Values shown are mean ± S.E. There was no statistically significant difference after application of topical verapamil. f Values shown are mean ± S.E. The difference between baseline and two hours after application of verapamil was statistically significant (P = .008).
of low-tension glaucoma and may have beneficial VOL.119,
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systolic velocity ■ diastolic velocity
0.063%
0.125%
0.25%
Verapamil Concentration
Fig. 3 (Netland and associates). Percent change of color Doppler ultrasound measurements of peak systolic and end diastolic velocities in the central retinal artery two hours after topical administration of 0.063%, 0.125%, and 0.25% verapamil (mean ± S.E.).
in systemic absorption of the drug from variable use of punctal occlusion or other factors. Topical administration of verapamil has been shown to decrease intraocular pressure in ocular hypertensive subjects.5,6 The effect on intraocular pressure is more pronounced in ocular hypertensive than in normotensive subjects.6 Our finding of re duced intraocular pressure after instillation of 0.125% and 0.25% verapamil eyedrops is consistent with these previous studies. Topical administration of ver apamil may result in up to 200-fold higher levels in the aqueous than those achieved with systemic ad ministration.16 However, higher concentrations of topical verapamil (1% or 2%) may not reduce intra ocular pressure,17 suggesting a dose-dependent, bi-
phasic response. Calcium channel blockers have multiple nonspecific actions at high concentrations, including blockade of potassium channels, inhibition of nucleoside transport, and low-affinity binding of neurotransmitter receptors.18 These nonspecific ac tions may produce effects that would not be predicted based solely on calcium channel blockade. Using color Doppler ultrasound imaging, we found a significantly reduced Pourcelot's ratio in the central retinal artery after topical administration of 0.125% verapamil, indicating decreased resistance in this vessel. There were also concurrent increases in end diastolic blood velocity and peak systolic velocity, but these were not statistically significant. Similar chang es of the resistive index have been observed using color Doppler ultrasound imaging after administra tion of timolol 0.5% eyedrops in the region of the central retinal and the posterior ciliary arteries.12 Our findings of reduced resistive index and increased blood velocity during the short time course of the testing indicate that these changes are likely a result of reduced distal vascular resistance and increased volumetric blood flow. An alternate explanation for these findings is development of a stenosis, which is unlikely because of the short time period of this study and the vasodilating action of this drug. We measured blood velocity because it is not currently possible to determine the cross-sectional area of ocular vessels and thereby calculate volumetric blood flow with color Doppler ultrasound imaging. Our correlation analysis showed that the changes of color Doppler ultrasound measurements in the central retinal artery after application of topical verapamil were not directly related to changes in intraocular pressure. In the range of pressures ob-
TABLE 3 COLOR DOPPLER ULTRASOUND MEASUREMENTS TWO HOURS AFTER APPLICATION OF TOPICAL 0.125% VERAPAMIL TEMPORAL POSTERIOR CILIARY ARTERY
Peak systolic velocity (cm/sec)* End diastolic velocity (cm/sec)* Pourcelot's ratio*
NASAL POSTERIOR CILIARY ARTERY
CENTRAL RETINAL VEIN
BASELINE
AFTER VERAPAMIL
BASELINE
AFTER VERAPAMIL
BASELINE
AFTER VERAPAMIL
9.88 ± 1.31 3.91 ± 0.79 0.63 ± 0.04
10.50 ± 1.83 4.03 ± 1.07 0.64 ± 0,05
8.63 ± 0.89 3.38 ± 0.39 0.61 ± 0.02
9.26 ± 0.97 3.24 ± 0.57 0.67 ± 0.02
3.75 ± 0.27 2.64 ± 0.25 0.32 ± 0.02
4.08 ± 0.31 3.09 ± 0.24 0.33 ± 0.06
"Values given are mean ± S.E There was no statistically significant difference after application of topical verapamil.
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served in these normal subjects, autoregulation of retinal blood flow would be expected to occur,19'20 unless the drug itself altered autoregulation. Under extreme conditions, for example, even normal eyes may not adequately compensate for intraocular pres sure between 30 and 39 mm Hg.19'21 However, in this study the mean baseline intraocular pressure was 13.9 mm Hg. The changes of resistive index we observed were likely caused by effects of verapamil on vascular resistance rather than by an indirect effect related to changes of intraocular pressure. After intravenous nicardipine in cats, Harino, Riva, and Petrig9 found enhanced optic nerve head but not retinal blood flow by using laser Doppler techniques. After application of topical verapamil, we found a decreased resistive index measured by color Doppler ultrasound in the central retinal artery, which supplies the retinal circulation. This apparent difference may not be inconsistent because different hemodynamic parameters are measured in vessels of differing calibers by these techniques. This difference may also have been from systemic rather than topical administration of the drug or, less likely, from interspecies differences and variability in the pharmaco logie activity between the two types of calcium channel blockers. Enhancement of retinal circulation may be relevant in glaucoma patients because gangli on cell bodies, which are markedly decreased in advanced glaucoma, are located in the inner retina and receive their blood supply from the retinal circulation. Our findings also indicate that topical calcium channel blockers may have applications in the treatment of retinal vascular abnormalities. Our color Doppler ultrasound measurements of the posterior ciliary arteries and central retinal vein showed no significant differences compared with baseline after application of topical verapamil. The blood supply to the optic nerve is primarily from branches of the short posterior ciliary arteries, and venous drainage occurs through the central retinal vein.22,23 Our finding of no significant changes mea sured by color Doppler ultrasound after application of topical verapamil could have several interpretations. First, an effect that was undetected in this study might be from a type 2 or false-negative error. Second, there may have been no change, because the short posterior ciliary arteries primarily supply the choroidal vasculature, which is a low-resistance vascular bed that may VOL.119, No. 6
not have the capacity to respond with further reduc tion of vascular resistance after verapamil treatment. Third, pharmacologie effects from dose or distribu tion of the drug may have prevented an effect on the choroidal circulation. In this regard, it is possible that after topical administration of verapamil optic nerve head blood flow could be enhanced because of high local concentration of drug in the posterior pole, with only a negligible effect on the blood flow in the posterior ciliary arteries if the choroidal circulation was largely unaffected by the drug. In this double-masked, prospective, randomized study in normal volunteers, we found no effect of a single drop of topical verapamil on blood pressure and heart rate. The intraocular pressure was significantly reduced after topical administration of verapamil. Using color Doppler ultrasound imaging, we found a decreased Pourcelot's ratio in the central retinal artery, indicating a reduced vascular resistance. Al though limitations of this study include a relatively small number of subjects and single-drop dosing, our findings indicate potentially beneficial ocular hemo dynamic effects from topical administration of the calcium channel blocker verapamil. Additional stud ies are needed to clarify the ocular effects of calcium channel blockers, which may have a role in the therapy of increased intraocular pressure and lowtension glaucoma. ACKNOWLEDGMENT
Statistical assistance was provided by Norman M. Aquino, M.D., Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, and Suguru Konno, M.D., Schepens Eye Research Institute, Boston, Massachu setts.
REFERENCES 1. Braunwald E. Mechanism of action of calcium-channelblocking agents. N Engl ] Med 1982;307:1618-27. 2. Monica ML, Hesse RJ, Messerli FH. The effect of a calciumchannel blocking agent on intraocular pressure [letter]. Am J Ophthalmol 1983;96:814. 3. Kelly SP, Walley TJ. Effect of the calcium antagonist nifedipine on intraocular pressure in normal subjects. Br J Ophthalmol 1988;72:216-8. 4. Schnell D. Response of intraocular pressure in normal subjects and glaucoma patients to single and repeated doses of the coronary drug adalat. In: Lochner W, Engel HJ, Lichtlen
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5. 6. 7. 8. 9. 10.
11. 12.
13. 14.
PR, editors. Second International Adalat Symposium. Berlin: Springer Verlag, 1975:290-302. Abelson MB, Gilbert CM, Smith LM. Sustained reduction of intraocular pressure in humans with the calcium channel blocker verapamil. Am J Ophthalmol 1988;105:155-9. Goyal JK, Khilnani G, Sharma DP, Singh J. The hypotensive effect of verapamil eye drops on ocular hypertension. Indian J Ophthalmol 1989;37:176-8. Netland PA, Chaturvedi N, Dreyer EB. Calcium channel blockers in the management of low-tension and open-angle glaucoma. Am J Ophthalmol 1993;115:608-13. Kitazawa Y, Shirai H, Go FJ. The effect of Ca2+-antagonist on visual field in low-tension glaucoma. Graefes Arch Clin Exp Ophthalmol 1989;227:408-12. Harino S, Riva CE, Petrig BL. Intravenous nicardipine in cats increases optic nerve head but not retinal blood flow. Invest Ophthalmol Vis Sei 1992;33:2885-90. Lieb WE, Cohen SM, Merton DA, Shields JA, Mitchell DG, Goldberg BB. Color Doppler imaging of the eye and orbit: technique and normal vascular anatomy. Arch Ophthalmol 1991;109:527-31. GuthoffRF, Berger RW, Winkler P, Helmke K, Chumbley LC. Doppler ultrasonography of the ophthalmic and central retinal vessels. Arch Ophthalmol 1991;109:532-6. Baxter GM, Williamson TH, McKillop G, Dutton GN. Color Doppler ultrasound of orbital and optic nerve blood flow: effects of posture and timolol 0.5%. Invest Ophthalmol Vis Sei 1992;33:604-10. Powis RL, Schwartz RA. Practical Doppler ultrasound for the clinician. Baltimore: Williams & Wilkins, 1991:171-2. Tamaki Y, Nagahara M, Yamashita H, Kikuchi M. Analysis of blood flow velocity of ophthalmic artery with color Doppler imaging, 1: studies on normal human eyes [in Japanese]. Nippon Ganka Gakkai Zasshi 1993;97:175-80.
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15. Harris A, Williamson TH, Shoemaker JA, Sergott RC, Spaeth GL, Katz JL, et al. Reproducibility of color Doppler imaging assessment of blood flow velocity in orbital vessels. J Glaucoma 1995. Forthcoming. 16. Ettl A, Daxer A, Hofmann U. Calcium channel blockers in the management of low-tension and open-angle glaucoma [letter]. Am J Ophthalmol 1993;116:778-80. 17. Beatty JF, Krupin T, Nichols PF, Becker B. Elevation of intraocular pressure by calcium channel blockers. Arch Ophthalmol 1984;102:1072-6. 18. Netland PA, Erickson KA. Calcium channel blockers in glaucoma management. Ophthalmol Clin North Am 1995. Forthcoming. 19. Riva CE, Grunwald JE, Petrig BL. Autoregulation of human retinal blood flow: an investigation with laser Doppler velocimetry. Invest Ophthalmol Vis Sei 1986;27:1706-12. 20. Harris A, Martin Bj, Shoemaker JA. Regulation of retinal blood flow during blood gas perturbation. J Glaucoma 1994;3(1 Suppl):S82~90. 21. Trible JR, Sergott RC, Spaeth GL, Wilson RP, Katz LJ, Moster MR, et al. Trabeculectomy is associated with retrobulbar hemodynamic changes: a color Doppler analysis. Oph thalmology 1994;101:340-51. 22. Hayreh SS. Blood supply of the optic nci ve head in health and disease. In: Lainbrou GN, Grève EL, editors. Ocular blood flow in glaucoma: means, methods and measurements. Amstelveen, The Netherlands: Kugler 6k Ghedini Publica tions, 1989:3-48. 23. Sugiyama K, Ciofri GA, Bacon DR, Van Buskirk EM. Optic nerve and peripapillary choroidal microvasculature in the primate. J Glaucoma 1994;3(1 Suppl):S45-54.
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• PURPOSE: To investigate the effect of topical administration of the calcium channel blocker verapamil on intraocular pressure and retrobulbar hemodynamics. • METHODS: In this randomized, prospective, double-masked study, we examined the effects of single-dose topical administration of verapamil in ten normal human volunteers by using color Doppler ultrasound imaging to measure hemodynamic parameters. Limitations of this study in clude single-dose application of verapamil and relatively small sample size. • RESULTS: N o systemic effect on heart rate or blood pressure was detected after administration of topical verapamil. The intraocular pressure signif icantly decreased compared with baseline two hours after topical 0.125% and 0.25% verapamil (P = .015 and . 0 4 0 , respectively). Pourcelot's ratio, an index of vascular resistance, measured in the central retinal artery was significantly reduced after topical application of 0.125% verapamil (P = .008). The change in Pourcelot's ratio primarily resulted from an increased end diastolic velocity in the central retinal artery. N o significant differenc es compared with baseline values were detected in the color Doppler ultrasound measurements of the Accepted for publication Dec. 27, 1994. From the Department of Ophthalmology, Massachusetts Eye and Ear Infirmary (Drs. Netland and Grosskreutz and Ms. Hart), and Schepens Eye Research Institute (Dr. Feke), Harvard Medical School, Boston, Massachusetts. This study was supported by the Massachusetts Lions Eye Research Fund, Northboro, Massachusetts, and CooperVision Pharmaceuticals, San Clémente, California. Reprint requests to Peter A. Netland, M.D., Department of Oph thalmology, Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 02114; fax: (617) 573-3707.
694
posterior ciliary arteries and the central retinal vein two hours after topically administered verapamil. • CONCLUSIONS: Topical administration of ver apamil decreases intraocular pressure and alters ocular hemodynamics, reducing the vascular re sistance index in the central retinal artery.
C
ALCIUM CHANNEL BLOCKERS ARE USED WIDELY for the treatment of cardiovascular disorders, including angina pectoris, cardiac arrhyth mias, systemic hypertension, and Raynaud's phenom enon. 1 These drugs reduce vascular resistance and help prevent vasospasm. The ocular effects of calcium channel blockers are of interest for their potential clinical role, in particular for the treatment of glauco ma patients. Systemic administration of calcium channel block ers in humans has generally resulted in a decreased intraocular pressure.2'4 In ocular hypertensive pa tients, topical verapamil causes a marked and sus tained reduction of intraocular pressure.5,6 This reduc tion of intraocular pressure suggests that verapamil might be used to treat hypertensive glaucoma. In addition to an effect on intraocular pressure, calcium channel blockers may have other beneficial effects in glaucoma because they inhibit vasospasm or enhance optic nerve blood flow, which may be particularly influential in low-tension glaucoma. In a previous retrospective cohort study, we found that the use of calcium channel blockers is associated with a slowed progression of low-tension glaucoma.7 Sustained improvement of visual fields has been described after administration of calcium channel
© AMERICAN JOURNAL OF OPHTHALMOLOGY ii9:694-7oo
JUNE 1995
blockers to patients with low-tension glaucoma.8 In an experimental study in cats, intravenous nicardipine caused an increased optic nerve head blood flow as measured by laser Doppler flowmetry.9 Color Doppler ultrasound imaging allows real-time B-scan ultrasound imaging of anatomic structures in the orbit with superimposed color-coded Doppler frequency shifts resulting from blood flow, allowing localization of vessels and measurement of vascular blood velocities.10'12 Using this noninvasive tech nique, we studied the effects of the topical calcium channel blocker verapamil on ocular circulation. We performed a prospective, double-masked, randomized study in normal human volunteers of the effect of single-dose topical verapamil on systemic blood pres sure and heart rate, intraocular pressure, and color Doppler ultrasound measurements of ocular blood vessels.
SUBJECTS AND METHODS VERAPAMIL OPHTHALMIC FORMULATION, PROVIDED BY
CooperVision Pharmaceuticals (San Clémente, Cali fornia), contained 0.003% benzalkonium chloride and 0.063%, 0.125%, or 0.25% verapamil. The placebo was identical to the vehicle of the verapamilcontaining solution. The drug and placebo drops were provided with coded labels so that neither the investi gators nor the patient had any knowledge of which eye was receiving the drug or which strength of drug. Ten volunteers in good health (seven women and three men with a mean ± S.D. age of 29 ± 6 years), who were not receiving any ocular or systemic medi cations, were recruited for the study. After informed consent was granted, baseline intraocular pressure, heart rate, systemic blood pressure, and color Doppler ultrasound values were measured. Eyedrops contain ing 0.063%, 0.125%, or 0.25% verapamil were in stilled randomly and in a double-masked fashion in one eye, and placebo was administered to the fellow eye. Intraocular pressure, heart rate, systemic blood pressure, and color Doppler ultrasound measurements were repeated two hours after instillation of eyedrops containing verapamil or placebo. Each subject was studied on three days, separated by at least one week between measurements, to obtain ten measurements for each of the three doses of verapamil. The order of VOL.119, No. 6
the three doses was randomized on the three days, with the examiners masked as to which doses were administered to each subject. Baseline measurements were repeated on each visit. Color Doppler ultrasound imaging was performed with a Siemens Quantum 2000 (Issaquah, Washing ton) using a 7.5-MHz transducer. The flow setting was for slow flow, and the sample volume was 1.2 mm3. The scan plane was horizontal through the optic nerve. The probe was carefully placed over the upper eyelid with minimal pressure. All color Doppler ultrasound measurements were performed by one of us (L.J.H.) without knowledge of which eyes had been treated with verapamil. Measurements of the peak systolic velocity, end diastolic velocity, and Pourcelot's ratio were performed in duplicate for the central retinal artery, nasal and temporal posterior ciliary arteries, and central retinal vein. Pourcelot's ratio was calculated as (peak systolic velocity minus end diastolic flow velocity) divided by peak systolic velocity.13 Pourcelot's ratio, which reflects vascular resistance, tends to be independent of the Doppler angle. The values of Pourcelot's ratio can vary from 0 to 1, with higher numbers indicating greater vascular resistance. The reproducibility of color Doppler ultrasound has been studied, with coefficients of variation in the ophthalmic artery ranging from 4% for the resistive index to 11% for peak systolic velocity.1415 Central retinal artery and vein measurements were performed within the less echogenic optic nerve shadow, ap proximately 2 mm posterior to the globe. Posterior ciliary vessels were located either temporally or nasal ly adjacent to the optic nerve shadow. We attempted to study the same site for subsequent measurements in each subject. The spectral measurement was anglecorrected to the longest axis obtainable, and Doppler angle did not vary on repeated measurements in the same subjects. The two-tailed paired t-test was used to evaluate the statistical significance of the change from baseline of each of the measured quantities and the differences in the changes between eyes receiving verapamil and eyes receiving placebo. In the central retinal artery measurements, the power of the test to determine a 12% change of Pourcelot's ratio was .57. Spearman correlation analysis was used to evaluate the relation ships between the changes in intraocular pressure and
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TABLE 1 SYSTEMIC HEART RATE AND BLOOD PRESSURE AFTER APPLICATION OF TOPICAL VERAPAMIL 0.063% VERAPAMIL
Heart rate (beats/min)* Systolic blood pressure (mm Hg)· Diastolic blood pressure (mm Hg)*
BASELINE
AFTER APPLICATION
72 ± 4 114 ± 2 69 ± 2
ne + 2
0.125% VERAPAMIL
0.25° » VERAPAMIL
BASELINE
AFTER APPLICATION
BASELINE
AFTER APPLICATION
68 ± 3 115i2 70 ± 2
70 ± 3 118 ± 2 72 ± 3
69 ± 3 115 ± 3 73 ± 3
68 ± 4 114 ± 2 73 ± 2
70 ± 4 71 ± 2
'Values shown are mean ± S.E. There was no statistically significant difference after topical application of verapamil.
the changes in the hemodynamic parameters. P < .05 were considered significant.
RESULTS THE
EFFECTS OF
TOPICALLY ADMINISTERED
0.063%,
0.125%, and 0.25% verapamil on systemic heart rate and blood pressure were studied (Table 1). Compared with baseline values, no statistically significant differ ences in heart rate were noted two hours after any of these doses of topical verapamil. Similarly, systolic and diastolic blood pressure measurements did not vary significantly two hours after application of topi cal verapamil. The mean baseline intraocular pressure was 13.9 ± 0.7 mm Hg (mean ± S.E.) for all placebo- and verapamil-treated eyes. As shown in Figure 1, the mean intraocular pressure was reduced compared with baseline in eyes treated with topical verapamil. This reduction of intraocular pressure was statistically significant after administration of 0.125% (P = .015) and 0.25% (P = .040) verapamil, but not after administration of 0.063% verapamil. The intraocular pressure of the fellow eyes, which were treated with placebo, did not vary significantly compared with baseline. Pourcelot's ratio, an index of vascular resistance, was measured two hours after topical administration of 0.063%, 0.125%, and 0.25% verapamil (Fig. 2). As shown in Table 2, the mean Pourcelot's ratio of the central retinal artery was significantly reduced com pared with baseline after administration of 0.125% verapamil (P = .008). At the higher 0.25% dose, Pourcelot's ratio was also decreased compared with baseline (Fig. 2), but this difference was not statisti 696
cally significant. There was no significant change in Pourcelot's ratio in the fellow placebo-treated eye at any of the examinations. On average, as shown in Table 2, systolic and diastolic velocities in the central retinal artery in creased after topical verapamil. As shown in Figure 3, the percent increase of the diastolic velocities was greater than that of the systolic velocities. The mean ± S.E. of the percent increase in diastolic velocity in the central retinal artery was 56% ± 25% after administration of 0.125% verapamil (P = .12) and
■ verapamil D placebo
o
0.063%
0.125%
0.25%
Verapamil Concentration
Fig. 1 (Netland and associates). Effect of topical verapa mil on intraocular pressure. The percent change of intraocular pressure (mean ± S.E.) from baseline is shown two hours after three different doses of verapamil for the treated eye and the fellow eye that received a placebo. The reduction of intraocular pressure was significant compared with baseline for the 0.125% and 0.25% doses of verapamil (P = .015 and .040, respec tively).
AMERICAN JOURNAL OF OPHTHALMOLOGY
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1995
baseline 2 hours
S. 0.70
0.063%
0.125%
0.25%
Verapamil Concentration
Fig. 2 (Netland and associates). Color Doppler ultra sound measurement of Pourcelot's ratio of the central retinal artery at baseline and two hours after topical administration of 0.063%, 0.125%, and 0.25% verapa mil (mean ± S.E.). The Pourcelot's ratio, a measure of vascular resistance, was significantly decreased com pared with baseline after 0.125% verapamil (P = .008).
35% ± 24% after administration of 0.25% verapamil (P = .16). In the eye that received placebo, the systolic velocity of the central retinal artery was increased by 15% ± 6% (P = .030) after 0.25% verapamil in the fellow eye, suggesting a possible crossover effect. Spearman correlation analysis showed no statistically significant relationships be tween the changes in intraocular pressure and the changes in systolic velocity, diastolic velocity, or Pourcelot's ratio in either eye at any of the concentra tions of verapamil. Color Doppler measurements were also performed in the temporal and nasal posterior ciliary arteries and in the central retinal vein. In these vessels, no significant differences compared with baseline meas urements were found in the mean peak systolic velocity, end diastolic velocity, or Pourcelot's ratio two hours after topical administration of 0.063%, 0.125%, or 0.25% verapamil. Color Doppler ultra sound measurements after administration of 0.125% verapamil are shown in Table 3.
DISCUSSION CALCIUM CHANNEL BLOCKERS SLOW THE PROGRESSION
effects on the visual fields of glaucoma patients.7,8 Possible mechanisms that may explain these effects include prevention of vasospasm or enhancement of ocular blood flow. Color Doppler ultrasound imaging is a noninvasive technique that may be used to assess the retrobulbar circulation.10'12 In this study, we exam ined the ocular and systemic effects of topically applied verapamil in a prospective, double-masked, randomized study in normal human volunteers. We found no effect on systemic blood pressure or heart rate but did find decreased intraocular pressure after administration of topical verapamil. Using color Doppler ultrasound imaging, we also demonstrated changes in ocular hemodynamics, with a reduction of the resistive index in the central retinal artery. Although systemic calcium channel blockers are known to decrease vascular resistance and reduce blood pressure, we found no change in systemic blood pressure or heart rate after topical administration of verapamil. Abelson, Gilbert, and Smith5 also found no change in blood pressure or heart rate after a single drop of topically applied verapamil. Goyal and associ ates,6 however, found decreased heart rate and blood pressure after two weeks of treatment with topical verapamil. This difference may be explained by the longer period of treatment or the higher baseline heart rates and blood pressures in the study by Goyal and associates. There may also have been differences TABLE 2 COLOR DOPPLER ULTRASOUND MEASUREMENTS IN THE CENTRAL RETINAL ARTERY TWO HOURS AFTER APPLICATION OF TOPICAL 0.125% VERAPAMIL
Peak systolic velocity (cm/sec)* End diastolic velocity (cm/sec)* Pourcelot's ratiof
BASELINE
AFTER APPLICATION OF VERAPAMIL
8.79 ± 0.44
8.87 ± 0.60
2.17 ± 0.34
2.78 ± 0.27
0.76 ± 0.03
0.67 ± 0.03
"Values shown are mean ± S.E. There was no statistically significant difference after application of topical verapamil. f Values shown are mean ± S.E. The difference between baseline and two hours after application of verapamil was statistically significant (P = .008).
of low-tension glaucoma and may have beneficial VOL.119,
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697
systolic velocity ■ diastolic velocity
0.063%
0.125%
0.25%
Verapamil Concentration
Fig. 3 (Netland and associates). Percent change of color Doppler ultrasound measurements of peak systolic and end diastolic velocities in the central retinal artery two hours after topical administration of 0.063%, 0.125%, and 0.25% verapamil (mean ± S.E.).
in systemic absorption of the drug from variable use of punctal occlusion or other factors. Topical administration of verapamil has been shown to decrease intraocular pressure in ocular hypertensive subjects.5,6 The effect on intraocular pressure is more pronounced in ocular hypertensive than in normotensive subjects.6 Our finding of re duced intraocular pressure after instillation of 0.125% and 0.25% verapamil eyedrops is consistent with these previous studies. Topical administration of ver apamil may result in up to 200-fold higher levels in the aqueous than those achieved with systemic ad ministration.16 However, higher concentrations of topical verapamil (1% or 2%) may not reduce intra ocular pressure,17 suggesting a dose-dependent, bi-
phasic response. Calcium channel blockers have multiple nonspecific actions at high concentrations, including blockade of potassium channels, inhibition of nucleoside transport, and low-affinity binding of neurotransmitter receptors.18 These nonspecific ac tions may produce effects that would not be predicted based solely on calcium channel blockade. Using color Doppler ultrasound imaging, we found a significantly reduced Pourcelot's ratio in the central retinal artery after topical administration of 0.125% verapamil, indicating decreased resistance in this vessel. There were also concurrent increases in end diastolic blood velocity and peak systolic velocity, but these were not statistically significant. Similar chang es of the resistive index have been observed using color Doppler ultrasound imaging after administra tion of timolol 0.5% eyedrops in the region of the central retinal and the posterior ciliary arteries.12 Our findings of reduced resistive index and increased blood velocity during the short time course of the testing indicate that these changes are likely a result of reduced distal vascular resistance and increased volumetric blood flow. An alternate explanation for these findings is development of a stenosis, which is unlikely because of the short time period of this study and the vasodilating action of this drug. We measured blood velocity because it is not currently possible to determine the cross-sectional area of ocular vessels and thereby calculate volumetric blood flow with color Doppler ultrasound imaging. Our correlation analysis showed that the changes of color Doppler ultrasound measurements in the central retinal artery after application of topical verapamil were not directly related to changes in intraocular pressure. In the range of pressures ob-
TABLE 3 COLOR DOPPLER ULTRASOUND MEASUREMENTS TWO HOURS AFTER APPLICATION OF TOPICAL 0.125% VERAPAMIL TEMPORAL POSTERIOR CILIARY ARTERY
Peak systolic velocity (cm/sec)* End diastolic velocity (cm/sec)* Pourcelot's ratio*
NASAL POSTERIOR CILIARY ARTERY
CENTRAL RETINAL VEIN
BASELINE
AFTER VERAPAMIL
BASELINE
AFTER VERAPAMIL
BASELINE
AFTER VERAPAMIL
9.88 ± 1.31 3.91 ± 0.79 0.63 ± 0.04
10.50 ± 1.83 4.03 ± 1.07 0.64 ± 0,05
8.63 ± 0.89 3.38 ± 0.39 0.61 ± 0.02
9.26 ± 0.97 3.24 ± 0.57 0.67 ± 0.02
3.75 ± 0.27 2.64 ± 0.25 0.32 ± 0.02
4.08 ± 0.31 3.09 ± 0.24 0.33 ± 0.06
"Values given are mean ± S.E There was no statistically significant difference after application of topical verapamil.
698
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1995
served in these normal subjects, autoregulation of retinal blood flow would be expected to occur,19'20 unless the drug itself altered autoregulation. Under extreme conditions, for example, even normal eyes may not adequately compensate for intraocular pres sure between 30 and 39 mm Hg.19'21 However, in this study the mean baseline intraocular pressure was 13.9 mm Hg. The changes of resistive index we observed were likely caused by effects of verapamil on vascular resistance rather than by an indirect effect related to changes of intraocular pressure. After intravenous nicardipine in cats, Harino, Riva, and Petrig9 found enhanced optic nerve head but not retinal blood flow by using laser Doppler techniques. After application of topical verapamil, we found a decreased resistive index measured by color Doppler ultrasound in the central retinal artery, which supplies the retinal circulation. This apparent difference may not be inconsistent because different hemodynamic parameters are measured in vessels of differing calibers by these techniques. This difference may also have been from systemic rather than topical administration of the drug or, less likely, from interspecies differences and variability in the pharmaco logie activity between the two types of calcium channel blockers. Enhancement of retinal circulation may be relevant in glaucoma patients because gangli on cell bodies, which are markedly decreased in advanced glaucoma, are located in the inner retina and receive their blood supply from the retinal circulation. Our findings also indicate that topical calcium channel blockers may have applications in the treatment of retinal vascular abnormalities. Our color Doppler ultrasound measurements of the posterior ciliary arteries and central retinal vein showed no significant differences compared with baseline after application of topical verapamil. The blood supply to the optic nerve is primarily from branches of the short posterior ciliary arteries, and venous drainage occurs through the central retinal vein.22,23 Our finding of no significant changes mea sured by color Doppler ultrasound after application of topical verapamil could have several interpretations. First, an effect that was undetected in this study might be from a type 2 or false-negative error. Second, there may have been no change, because the short posterior ciliary arteries primarily supply the choroidal vasculature, which is a low-resistance vascular bed that may VOL.119, No. 6
not have the capacity to respond with further reduc tion of vascular resistance after verapamil treatment. Third, pharmacologie effects from dose or distribu tion of the drug may have prevented an effect on the choroidal circulation. In this regard, it is possible that after topical administration of verapamil optic nerve head blood flow could be enhanced because of high local concentration of drug in the posterior pole, with only a negligible effect on the blood flow in the posterior ciliary arteries if the choroidal circulation was largely unaffected by the drug. In this double-masked, prospective, randomized study in normal volunteers, we found no effect of a single drop of topical verapamil on blood pressure and heart rate. The intraocular pressure was significantly reduced after topical administration of verapamil. Using color Doppler ultrasound imaging, we found a decreased Pourcelot's ratio in the central retinal artery, indicating a reduced vascular resistance. Al though limitations of this study include a relatively small number of subjects and single-drop dosing, our findings indicate potentially beneficial ocular hemo dynamic effects from topical administration of the calcium channel blocker verapamil. Additional stud ies are needed to clarify the ocular effects of calcium channel blockers, which may have a role in the therapy of increased intraocular pressure and lowtension glaucoma. ACKNOWLEDGMENT
Statistical assistance was provided by Norman M. Aquino, M.D., Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, and Suguru Konno, M.D., Schepens Eye Research Institute, Boston, Massachu setts.
REFERENCES 1. Braunwald E. Mechanism of action of calcium-channelblocking agents. N Engl ] Med 1982;307:1618-27. 2. Monica ML, Hesse RJ, Messerli FH. The effect of a calciumchannel blocking agent on intraocular pressure [letter]. Am J Ophthalmol 1983;96:814. 3. Kelly SP, Walley TJ. Effect of the calcium antagonist nifedipine on intraocular pressure in normal subjects. Br J Ophthalmol 1988;72:216-8. 4. Schnell D. Response of intraocular pressure in normal subjects and glaucoma patients to single and repeated doses of the coronary drug adalat. In: Lochner W, Engel HJ, Lichtlen
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PR, editors. Second International Adalat Symposium. Berlin: Springer Verlag, 1975:290-302. Abelson MB, Gilbert CM, Smith LM. Sustained reduction of intraocular pressure in humans with the calcium channel blocker verapamil. Am J Ophthalmol 1988;105:155-9. Goyal JK, Khilnani G, Sharma DP, Singh J. The hypotensive effect of verapamil eye drops on ocular hypertension. Indian J Ophthalmol 1989;37:176-8. Netland PA, Chaturvedi N, Dreyer EB. Calcium channel blockers in the management of low-tension and open-angle glaucoma. Am J Ophthalmol 1993;115:608-13. Kitazawa Y, Shirai H, Go FJ. The effect of Ca2+-antagonist on visual field in low-tension glaucoma. Graefes Arch Clin Exp Ophthalmol 1989;227:408-12. Harino S, Riva CE, Petrig BL. Intravenous nicardipine in cats increases optic nerve head but not retinal blood flow. Invest Ophthalmol Vis Sei 1992;33:2885-90. Lieb WE, Cohen SM, Merton DA, Shields JA, Mitchell DG, Goldberg BB. Color Doppler imaging of the eye and orbit: technique and normal vascular anatomy. Arch Ophthalmol 1991;109:527-31. GuthoffRF, Berger RW, Winkler P, Helmke K, Chumbley LC. Doppler ultrasonography of the ophthalmic and central retinal vessels. Arch Ophthalmol 1991;109:532-6. Baxter GM, Williamson TH, McKillop G, Dutton GN. Color Doppler ultrasound of orbital and optic nerve blood flow: effects of posture and timolol 0.5%. Invest Ophthalmol Vis Sei 1992;33:604-10. Powis RL, Schwartz RA. Practical Doppler ultrasound for the clinician. Baltimore: Williams & Wilkins, 1991:171-2. Tamaki Y, Nagahara M, Yamashita H, Kikuchi M. Analysis of blood flow velocity of ophthalmic artery with color Doppler imaging, 1: studies on normal human eyes [in Japanese]. Nippon Ganka Gakkai Zasshi 1993;97:175-80.
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15. Harris A, Williamson TH, Shoemaker JA, Sergott RC, Spaeth GL, Katz JL, et al. Reproducibility of color Doppler imaging assessment of blood flow velocity in orbital vessels. J Glaucoma 1995. Forthcoming. 16. Ettl A, Daxer A, Hofmann U. Calcium channel blockers in the management of low-tension and open-angle glaucoma [letter]. Am J Ophthalmol 1993;116:778-80. 17. Beatty JF, Krupin T, Nichols PF, Becker B. Elevation of intraocular pressure by calcium channel blockers. Arch Ophthalmol 1984;102:1072-6. 18. Netland PA, Erickson KA. Calcium channel blockers in glaucoma management. Ophthalmol Clin North Am 1995. Forthcoming. 19. Riva CE, Grunwald JE, Petrig BL. Autoregulation of human retinal blood flow: an investigation with laser Doppler velocimetry. Invest Ophthalmol Vis Sei 1986;27:1706-12. 20. Harris A, Martin Bj, Shoemaker JA. Regulation of retinal blood flow during blood gas perturbation. J Glaucoma 1994;3(1 Suppl):S82~90. 21. Trible JR, Sergott RC, Spaeth GL, Wilson RP, Katz LJ, Moster MR, et al. Trabeculectomy is associated with retrobulbar hemodynamic changes: a color Doppler analysis. Oph thalmology 1994;101:340-51. 22. Hayreh SS. Blood supply of the optic nci ve head in health and disease. In: Lainbrou GN, Grève EL, editors. Ocular blood flow in glaucoma: means, methods and measurements. Amstelveen, The Netherlands: Kugler 6k Ghedini Publica tions, 1989:3-48. 23. Sugiyama K, Ciofri GA, Bacon DR, Van Buskirk EM. Optic nerve and peripapillary choroidal microvasculature in the primate. J Glaucoma 1994;3(1 Suppl):S45-54.
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