The Effect of Systemic Nitric Oxide-synthase Inhibition on Ocular Fundus Pulsations in Man

Exp. Eye Res. (1997) 64, 305–312

The Effect of Systemic Nitric Oxide-synthase Inhibition on Ocular Fundus Pulsations in Man L E O P O L D S C H M E T T E R ERa,b*, K U R T K R E J C Ya,c, J O H A N N E S K A S T N ERa, M I C H A E L W O L ZTa, G H A Z A L E H G O U Y Aa, O L I V E R F I N D Ld, F R A N Z L E X E Rb, H E L E N E B R E I T E N E D ERa, A D O L F F R I E D R I C H F E R C H ERb    H A N S-G E O R G E I C H L ERa a

Department of Clinical Pharmacology, b Institute of Medical Physics, c Institute of Pharmacology and d Department of Ophthalmology B University of Vienna, Vienna, Austria (Received Lund 14 May 1996 and accepted in revised form 10 July 1996 ) There is experimental evidence that endothelium derived nitric oxide is involved in the regulation of ocular vascular tone. The purpose of this study was to investigate the effects of NO-synthase inhibition by N-monomethyl--arginine (-NMMA) on ocular fundus pulsations in young healthy volunteers. Three milligrams per kilograms -NMMA were administered i.v. over 5 minutes. Protocol 1 : Measurements of blood pressure, pulse rate, fundus pulsation amplitude, NO-exhalation, and cardiac output were performed at baseline and 10, 30, 60, 90, 150, and 300 minutes after -NMMA infusion (n ¯ 8). Fundus pulsation amplitude, which has been shown to estimate the pulsatile component of the choroidal blood flow, was recorded with a recently developed laser interferometer. Protocol 2 : Measurements of blood pressure, pulse rate, fundus pulsation amplitude, NO-exhalation, and blood flow velocity in the ophthalmic artery were performed in a randomized, placebo controlled cross over study (n ¯ 10). Ten minutes after -NMMA administration fundus pulsation amplitude decreased by 23³2 % (protocol 1) and 19³1 % (protocol 2, P ! 0±01 each), cardiac output by 12³2 % (P ! 0±01), and exhaled NO by 55³6 % (protocol 1) and 41³6 % (protocol 2, P ! 0±01 each). All parameters returned to baseline values within the 300 minutes observation period, with a faster recovery of fundus pulsation amplitude than of cardiac output and exhaled NO. Blood pressure, pulse rate, and ophthalmic artery blood flow velocity showed only minor changes during and after administration of -NMMA. Our results suggest that systemic NO-synthase inhibition reduces pulsatile choroidal and most likely total choroidal blood flow in humans. The recovery of vascular tone in choroidal vessels seems to be different from the cardiovascular response. Our findings indicate that reduced fundus pulsations after -NMMA are caused by systemic factors as well as by local reactions of the choroidal vasculature. # 1997 Academic Press Limited Key words : nitric oxide ; fundus pulsations ; choroidal circulation ; human.

1. Introduction Nitric oxide (NO), which is synthesized in the vascular endothelium from the guanidino nitrogen atoms of arginine (Palmer et al., 1988), has been shown to control the resting tone in different vascular beds (Gardiner et al., 1990). The introduction of inhibitors of NO-synthase such as N-monomethyl--arginine (NMMA) has enabled the investigation of the physiological role of NO in the regulation of blood flow and blood pressure (Palmer and Moncada, 1989 ; Mayer et al., 1989 ; Rees et al., 1990). Animal studies demonstrated that systemic infusion of -NMMA causes a rise in blood pressure and a vasoconstriction in different vascular beds (Rees, Palmer and Moncada, 1989 ; Aisaka et al., 1989 ; Whittle et al., 1989). In humans, systemic infusion of -NMMA increased blood pressure and systemic vascular resistance and decreased cardiac output and stroke volume (Haynes et al., 1993 ; Stamler et al., 1994). * Corresponding author : Dr L. Schmetterer, Institute of Medical Physics, Wa$ hringer Straße 13, A-1090 Vienna, Austria.

0014–4835}97}030305­08 $25.00}0}ey960213

Although the effect of NO on vascular tone in ocular vessels has not yet been thoroughly studied, in vitro and animal studies suggest the physiologic importance of NO-synthesis to maintain an appropriate blood flow. The endothelium-dependent regulation of vascular tone has been demonstrated in pig (Yoo, Tschudi and Lu$ scher, 1991) and human (Haefliger, Flammer and Lu$ scher, 1992) isolated ophthalmic artery. Further investigations have demonstrated the physiologic importance of NO in the regulation of the ophthalmic microcirculation in perfused porcine eye (Meyer, Flammer and Lu$ scher, 1993a). Systemic infusion of the nitric oxide inhibitor nitro -arginine methylester (-NAME) significantly reduced choroid, ciliary body, and iris, but not retinal blood flow in beagle dogs (Deussen, Sonntag and Vogel, 1993). These results were confirmed in cats, in which systemic inhibition of NO-synthase by Nω-nitro--arginine (NNL-A) dosedependently decreased choroidal blood flow (Mann et al., 1995). Altered levels of local NO production have also been discussed in the pathophysiology of ocular diseases : Impaired endothelial formation of NO has been # 1997 Academic Press Limited

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suggested to contribute to the reduced ocular blood flow during retrobulbar anesthesia (Meyer, Flammer and Lu$ scher, 1993b). On the other hand, investigations in rats have shown that the hemodynamic changes associated with endotoxin-induced uveitis, an experimental model for intraocular inflammation, are mediated by an increased NO-synthesis (Mandai et al., 1994 ; Tilton et al., 1994). However, the influence of NO-synthase inhibition on ocular circulation has not been studied in humans. The aim of the present study was therefore to investigate the effect of systemic infusion of -NMMA on ocular blood flow parameters in young healthy volunteers. Laser interferometric measurement of local fundus pulsations (Schmetterer et al., 1995) as well as Doppler ultrasonography of the ophthalmic artery (OA) was done. The fundus pulsation amplitude (FPA), which equals the distance change between cornea and retina during the cardiac cycle, is an appropriate measure to characterize drug effects on local pulsatile oscular blood flow (Schmetterer et al., 1996a,b). These results were compared with non-invasive systemic hemodynamic measurements (cardiac output, blood pressure, pulse rate, systemic vascular resistance), and related to the degree of NO-synthase inhibition, as estimated by measurement of exhaled NO. 2. Materials and Methods Subjects Two consecutive studies, an open pilot study (protocol 1) and a randomized double-blind 2-way cross over study (protocol 2) were performed. The studies were approved by the Ethics Committee of Vienna University School of Medicine. Eight healthy male volunteers participated in study 1 (age range 21–31, mean 25±1³3±2 ..), and 10 healthy male volunteers in study 2 (age range 23–33, mean 26±6³3±1 ..). Written informed consent to participate was obtained. Each subject passed a screening examination, including physical examination and medical history, hematological status, clinical chemistry, hepatitis A, B, C and HIV-serology, and urine analysis, to determine health status. Subjects were excluded if any abnormality was found as part of the pretreatment screening unless the investigator considered an abnormality to be clinically irrelevant. Furthermore, an ophthalmic examination including slit lamp biomicroscopy, indirect funduscopy, tonometry and determination of refraction and visual acuity was performed. Inclusion criteria were normal ophthalmic findings and ametropy ! 3 dpt. Study Protocol Protocol 1 After an overnight fast, all subjects rested for at least 20 minutes in a sitting position to establish a stable baseline. After the baseline measure-

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ments of fundus pulsation amplitude (FPA), cardiac output (CO), blood pressure (BP), pulse rate (PR), and exhaled NO, 3 mg kg−" -NMMA (Clinalfa, La$ ufelfingen, Switzerland) were administered i.v. over 5 minutes. Measurements of hemodynamic parameters and of exhaled NO were performed in a predetermined order (FPA, CO, BP, PR, and exhaled NO) 10, 30, 60, 90, 150, and 300 minutes after the end of -NMMA infusion. The subjects remained comfortably in a sitting position until three hours after the end of drug administration under continuous ECGand pulse rate-monitoring. The -NMMA dose chosen in the pilot study (Protocol 1) was appropriate to significantly decrease FPA vs. baseline. A randomized placebo controlled study was added and was focused on ocular hemodynamics. Protocol 2 After an overnight fast, all subjects rested for at least 20 minutes in a sitting position to establish a stable baseline. After the baseline measurements of fundus pulsation amplitude (FPA), mean flow velocity (MFV) and resistive index (RI) in the ophthalmic artery, blood pressure (BP), pulse rate (PR), and exhaled NO, 3 mg kg−" -NMMA or placebo was administered i.v. over 5 minutes in a randomized double-blind 2-way cross over design. The washoutperiod between the two study days was at least 7 days. Measurements of hemodynamic parameters and of exhaled NO were performed in a predetermined order (FPA, MFV and RI in the OA, BP, PR, and exhaled NO) 5, 35, and 90 minutes after the end of -NMMA infusion. Methods of Evaluation Laserinterferometric measurement of fundus pulsations The method is based on the interference of the reflected beams from the cornea and retina, when the eye is illuminated with coherent light. Using a single mode laser diode the distance changes between cornea and retina during the cardiac cycle can be assessed with high accuracy (Schmetterer et al., 1995). These distance changes depend on the pulsatile inflow of arterial blood into the eye. During systole the blood volume in the eye increases, leading to a reduction in the distance between cornea and retina and a slightly increased intraocular pressure. The latter phenomenon has been used for the measurement of pulsatile ocular blood flow (POBF) with the Langham pneumatic tonometer (Langham et al., 1989). In contrast, laser interferometric measurement of fundus pulsations enables non-contactile estimation of POBF with high transversal resolution (Schmetterer et al., 1996a,b). In order to specifically assess -NMMAinduced effects on choroidal circulation, we performed measurements in the macula, where the retina lacks vasculature. FPAs were calculated as the maximum distance change between cornea and retina during the cardiac cycle.

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Doppler ultrasonic measurement of cardiac output Cardiac output was assessed by Doppler ultrasound at the aortic valve using a 3,25 Mhz probe. Cardiac output was calculated by cross sectional area measurement of the aortic valve and determination of the velocity distribution profile. This method has been shown to be primarily suitable for measuring cardiac output changes within a subject rather than comparing absolute values of cardiac output between different subjects (Coates, 1990).

Alto, CA, U.S.A.). The systemic vascular resistance was calculated as SVR ¯ MAP}CO (Protocol 1).

Doppler ultrasonic measurement of blood flow velocity in the ophthalmic artery. In the ophthalmic artery the peak systolic flow velocity (PSV) and end diastolic flow velocity (EDV) was assessed with Duplex imaging using a 7±5 MHz color Doppler probe (Lieb et al., 1991 ; Guthoff et al., 1991). Mean flow velocity (MFV) was measured as the time mean of the spectral outline. From these parameters the resistive index was calculated as : RI ¯ (PSV®EDV)}PSV. The ophthalmic artery (OA) was measured anteriorly, at the point where it crosses the optic nerve. The sample volume marker was placed approximately 25 mm posterior to the globe. A coupling gel was placed on the upper lid of the closed right eye and the probe was positioned with minimal pressure. Measurement of exhaled NO. Exhaled nitric oxide was measured with a chemoluminescence detector (Monitor Labs Inc., Nitrogen oxides analyser, Model 8840, U.S.A.) connected to a strip-chart recorder. The instrument was calibrated with certified gases (300 ppb NO in N , AGA, Vienna, Austria), diluted by # precision flow meters. A baseline signal was obtained with pure nitrogen. Subjects were instructed to fully inflate their lungs, hold their breath for 10 seconds, and exhale for 10 seconds into a teflon tube. 1000 ml min−" of the exhaled air was allowed to enter the inlet port. Three consecutive readings were made at each measurement point under nasal occlusion. The end-expiratory values from the strip recorder readings were used for analysis to assure that inspired NO from the ambient air does not distort the results (Jilma et al., 1996). This method of quantifying the degree of endogenous NO-synthesis has already been used previously (Kharitonov et al., 1994). Noninvasive measurement of systemic hemodynamics. Systolic and diastolic blood pressure (SBP, DBP) were measured on the upper arm by an automated oscillometric device. Pulse pressure amplitude (PPA) was calculated as SBP-DBP, mean arterial pressure (MAP) was calculated at 1}3 SBP­2}3 DBP. Pulse rate (PR) was automatically recorded from a finger pulseoxymetric device, ECG was taken from a standard device (HP-CMS patient monitor, Hewlett Packard, Palo

Statistical Analysis Statistical analysis was done with the CSS Statistica2 software package (StatSoft Inc., Tulsa, OK, U.S.A.). Standard deviation and standard error of the mean were calculated. For protocol 1 changes in hemodynamic parameters were analysed with Friedman ANOVA, and the Wilcoxon-signed rank test for post hoc comparison using the absolute values before and after -NMMA infusion. Data are presented as percent of baseline (%). For protocol 2 changes in hemodynamic parameters were analysed with 2-way repeated measure ANOVA using the absolute values of -NMMA and placebo study days, respectively. Post hoc comparisons between treatment groups were done with paired t test at individual time points. A P value of ! 0±05 was considered significant. For data description values are given as means³...

3. Results Protocol 1 The effects of -NMMA on hemodynamic parameters and exhaled NO 10 minutes after the end of infusion are summarized in Table I. -NMMA caused a maximum decrease in exhaled NO of 55³6 %, in CO of 12³2 %, and in FPA of 23³2 % and an increase in SVR of 16³3 %, (each P ! 0±01). T I Change in hemodynamic parameters before and 10 minutes after the end of a 5-minute infusion of 3 mg kg−" LNMMA. Values are expressed as means³1 standard deviation. Asterisks indicate significant differences (P ! 0±05 ) from baseline as calculated by Wilcoxon’s-signed rank test (n ¯ 8, protocol 1 ). Baseline

10 min after -NMMA

Fundus pulsation amplitude 3±9³1±3 3±0³1±2* [µm] Cardiac output [l min−"] 6±8³2±1 6±0³2±1* Exhaled NO [ppb] 44±7³27±1 20±2³19±3* Systolic blood pressure 125±0³12±9 121±3³4±5 [mm Hg] Diastolic blood pressure 65±0³7±9 70±6³9±3 [mm Hg] Pulse pressure amplitude 60±0³7±9 50±6³8±1 [mm Hg] Mean arterial pressure 85±0³6±5 87±5³7±0 [mm Hg] Pulse rate [l min−"] 69±5³9±6 65±2³10±6 Systemic vascular resistance 1013³295 1176³380* [dyn.s.cm−&]

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F. 1. Time course of the effect of systemic infusion of NMMA (3 mg kg−") on fundus pulsation amplitude (+), cardiac output (E), and exhaled NO (y). The data are expressed as percent of baseline ; time is measured from the end of the 5-minutes infusion period. Error bars represent the standard error of the mean (n ¯ 8, protocol 1).

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Fundus pulsation amplitude 3±6³1±4 3±5³1±4 [µm] Mean flow velocity 15±9³6±2 16±5³5±1 [cm s−"] Resistive index 0±88³0±03 0±89³0±02 Exhaled NO [ppb] 74±7³41±4 75±3³45±6 Systolic blood pressure 113±9³10±2 111±4³10±1 [mm Hg] Diastolic blood pressure 60±4³9±4 56±4³9±1 [mm Hg] Pulse pressure amplitude 53±5³9±9 55±0³9±8 [mm Hg] Mean arterial pressure 79±0³8±9 75±7³8±0 [mm Hg] Pulse rate [1 min−"] 61±6³13±8 57±9³13±1

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induced by -NMMA was observed 10 minutes after the end of infusion. All parameters returned to near baseline values after 300 minutes. Blood pressure and PR were unchanged during the observation period. In Fig. 2, the -NMMA-induced changes in exhaled NO and CO (upper panel), and exhaled NO and FPA (lower panel) are plotted in time sequence. Displaying data of Fig. 1 as effect on one variable against effect on another variable reveals the association or dissociation in time between the courses of these variables. The observed clockwise hysteresis loop comparing the time course of decrease in exhaled NO and FPA is indicative of a separation between degree of systemic NOsynthase inhibition and fundus pulsation amplitude. Protocol 2

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F. 2. Hysteresis loops : Changes from baseline after NMMA infusion for cardiac output and exhaled nitric oxide (upper panel), and for fundus pulsation amplitude and exhaled nitric oxide (lower panel) after systemic infusion of 3 mg kg−" -NMMA are plotted for each measurement point (1 : baseline, 2 : 10 min, 3 : 30 min, 4 : 60 min, 5 : 90 min, 6 : 150 min, 7 : 300 min after the end of infusion). Results are presented as percent of baseline. Consecutive measurement points are connected with arrows. Error bars represent the standard error of the mean (n ¯ 8, protocol 1).

The time course of exhaled NO, CO and FPA during the observation period is graphically shown in Fig. 1. The maximum effect on FPA, CO, and exhaled NO

Baseline values of the parameters under study are given in Table II ; no significant pretreatment differences were observed between the study days. Figure 3 summarizes the time course of ocular hemodynamic parameters and exhaled NO. FPA and exhaled NO were significantly different after -NMMA as compared to baseline. Five minutes after administration FPA decreased by 19³1 % and exhaled NO decreased by 41³6 % (each P ! 0±01). Parameters calculated from Doppler ultrasonographic data were not significantly different v. placebo. Only at the 5 minutes time point PSV significantly decreased by 11³3 % (P ! 0±05). MFV slightly decreased ; the other parameters were not affected by infusion of -NMMA. The time course of systemic parameters following infusion of -NMMA or placebo is shown in Fig. 4. The parameters did not significantly differ during -NMMA administration as compared to placebo. Five minutes after -NMMA diastolic blood pressure significantly increased by 13³5 % (P ! 0±05).

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F. 3. Time course of the effect of systemic infusion of -NMMA (3 mg kg−" ; solid lines) or placebo (..........) on fundus pulsation amplitudes (FPA), peak systolic flow velocity (PSV), end diastolic flow velocity (EDV), mean flow velocity (MFV), resistive index (RI) in the ophthalmic artery and exhaled NO (NO). The data are expressed as percent of baseline ; time is measured from the end of the 5-minutes infusion period. Error bars represent the standard error of the mean (n ¯ 10, protocol 2). Asterisks indicate significant results as analysed by repeated measure ANOVA, plus symbols indicate significant results as analysed by post-hoc comparisons.

4. Discussion Our results indicate that systemic inhibition of NOsynthase reduces pulsatile choroidal blood flow in man. In both studies 3 mg kg−" -NMMA caused a significant decrease in FPA of 23 % and 19 %, respectively. This confirms previous findings in dogs

(Deussen et al., 1993) and in cats (Mann et al., 1995), which have shown a decrease in choroidal blood flow following the administration of NO-synthase inhibitors. This is of major importance for the ocular circulation since animal experiments indicate that 85 % of the blood volume in the eye circulates in the choroid (Bill, 1975).

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F. 4. Time course of the effect of systemic infusion of -NMMA (3 mg kg−", solid lines) or placebo (dotted lines) on systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure amplitude (PPA), and pulse rate. The data are expressed as percent of baseline ; time is measured from the end of the 5-minutes infusion period. Error bars represent the standard error of the mean (n ¯ 10, protocol 2). Plus symbols indicate significant results as analysed by post-hoc comparisons.

Laser interferometric measurement of fundus pulsations does not directly assess ocular blood flow but yields an indirect measure of pulsatile blood flow in the choroid (Schmetterer et al., 1996a,b). Changes in systemic parameters (BP and CO), as well as local changes in the ocular vessels might influence our results. A blood pressure increase was only observed immediately after administration of -NMMA (Fig. 4). This effect was more pronounced on DBP than on SBP and therefore PPA was slightly reduced. In the pilot study the increase in DBP 10 minutes after administration was 8 %, but did not reach statistical significance. The rapid disappearance of this blood pressure effect argues for very rapid systemic counterregulatory mechanisms following NO-synthase inhibition. The effect on SVR and CO was long-lasting with a 16 % and 12 % change 10 minutes after administration, respectively. Both the decrease in CO and the decrease in PPA might partially be responsible for the effects on FPA. However, in the OA a decrease in PSV was only observed immediately after the end of -NMMA infusion. Changes in blood flow velocity cannot necess-

arily be extrapolated to changes in blood flow (Kontos, 1989). Nevertheless it is very likely that we could have detected changes as small as approximately 10 % since the short-term variability of the method is low (Schmetterer et al., 1996b ; Harris et al., 1995). Hence our results obtained in the OA argue that local reactions to -NMMA as well as systemic factors contribute to the decrease in FPA. Moreover, the Doppler ultrasonic data argue that flow pulsatility was not significantly changed in the OA. An effect on smaller vessels in the choroid should increase vascular resistance and therefore flow pulsatility. It might therefore well be that the effect on FPA might underestimates the effect on total choroidal blood flow. Figure 2 (upper panel) shows that the effect of NMMA on the time course of exhaled NO and CO is similar. This indicates that the decrease of CO is closely and consistently related to the degree of systemic NOsynthase inhibition. In contrast, a clockwise hysteresis loop was observed between reduction in FPA and exhaled NO (Fig. 2, lower panel). This is indicative of a faster recovery in choroidal blood flow than in CO. During the first 60 minutes of the observation period

EFFECT OF NO ON OCULAR FUNDUS PULSATIONS

exhaled NO remained consistently at 45–50 % of baseline, whereas FPA increased from 77 % to 89 % of the pretreatment value during the same period. The more pronounced decrease and faster recovery of choroidal blood flow, as compared to cardiac output, indicate that choroidal blood flow after NO-synthase inhibition is not merely a function of systemic hemodynamic changes, but argue for a local reaction of choroidal vasculature. The rapid return to baseline level in FPA could be attributed to counterregulating mechanisms. Several endothelium derived factors such as prostacyclin, endothelin-1, thromboxane A and prostaglandin H # # have been shown to contribute to the regulation of ocular vascular tone (Haefliger et al., 1994). On the other hand it has to be taken into consideration that measurement of exhaled NO is not a direct quantification of endogenous NO production. Especially it is likely that exhaled NO is an inappropriate index of NOsynthase inhibition at the level of ocular circulation. Given the fact that the choroid has a very high perfusion level the effective concentration of -NMMA administered as a bolus may be different from that expressed in exhaled air. However, since NO is not accessible for a direct measurement, we used exhaled NO as a marker of NO-production. This method has already been used to quantify sex-differences in NOproduction as well as changes following administration of NO-synthase inhibitors (Kharitonov et al., 1994 ; Krejcy et al., 1995) and -arginine (Kharitonov et al., 1995). Additionally concerns have been raised regarding the specificity of -NMMA as an inhibitor of NOsynthase (Iadecola et al., 1994). We cannot exclude that -NMMA exerts other effects on ocular metabolism. Nevertheless, the effectiveness of -NMMA for NO-synthase inhibition in our study has been evidenced by the observed decrease in exhaled NO. Our results cannot be extrapolated to the retinal circulation, where both myogenic and metabolic factors strongly influence regulation of vascular tone (Bill, 1975). The reduction of blood flow following systemic NO-synthase inhibition in beagle dogs was much smaller in the retina than in the choroid (Deussen et al., 1993). However, local perivascular microinjections of nitro -arginine methylester (NAME) into the retina of miniature pigs suggest a role for NO in the regulation of vascular tone (Donati et al., 1994). In summary, we demonstrated that systemic NOsynthase inhibition reduces pulsatile choroidal and most likely total choroidal blood flow in humans. Together with other endothelium-derived relaxing factors (Haefliger et al., 1994) NO appears essential to maintain the high level of choroidal perfusion. It may be speculated that endothelial cell dysfunction in diabetes and hypertension, resulting in insufficient NO-synthesis, may contribute to regional ocular perfusion defects.

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Acknowledgements Excellent technical support from Brigitte Monitzer RN is acknowledged. Supported by the Jubilaumsfonds der Nationalbank (grant Nr. 4733).

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