Role of Nitric Oxide in Optic Nerve Head Blood Flow Regulation during Isometric Exercise in Healthy Humans MATERIALS AND METHODS.

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1 Clinical Trials Role of Nitric Oxide in Optic Nerve Head Blood Flow Regulation during Isometric Exercise in Healthy Humans Doreen Schmidl, 1 Agnes Boltz, 1,2 Semira Kaya, 1 Michael Lasta, 1 Berthold Pemp, 3 Gabriele Fuchsjager-Mayrl, 3 Anton Hommer, 4 Gerhard Garhofer, 1 and Leopold Schmetterer 1,2 PURPOSE. We determined whether administration of a nitric oxide synthase (NOS) inhibitor alters optic nerve head blood flow (ONHBF) regulation during isometric exercise in healthy subjects. METHODS. Our study was done in a randomized, placebocontrolled, double-masked, three-way crossover design. A total of 18 healthy subjects was randomized to receive either placebo, phenylephrine, or an inhibitor of NOS (L-NMMA) on three different study days. ONHBF was measured with laser Doppler flowmetry while the study participants performed isometric exercise (squatting). This was done before drug administration and during infusion of the study drugs. Mean arterial pressure (MAP) and IOP were measured noninvasively, and ocular perfusion pressure (OPP) was calculated as 2/3 MAP IOP. RESULTS. The response in ONHBF to isometric exercise was less pronounced than the response in OPP, indicating for some autoregulatory capacity in the ONH. Administration of L- NMMA significantly decreased ONHBF at rest (P < 0.01). In contrast, inhibition of NOS did not alter the pressure flow relationship in the ONH during an experimental increase in OPP compared to phenylephrine and placebo (P ¼ 0.37 between groups). CONCLUSIONS. The data of our study support previous findings that ONHBF is autoregulated during an experimental increase in OPP. Nitric oxide has an important role in basal ONHBF regulation, but seems not to be involved in the autoregulatory response during an increase in OPP induced by isometric exercise. (ClinicalTrials.gov number, NCT ) (Invest Ophthalmol Vis Sci. 2013;54: ) DOI: / iovs Several studies have shown that retinal blood flow is autoregulated in response to experimental changes in ocular perfusion pressure (OPP). 1 6 There also is evidence From the 1 Department of Clinical Pharmacology, the 2 Center for Medical Physics and Biomedical Engineering, and the 3 Department of Ophthalmology, Medical University of Vienna, Vienna, Austria; and the 4 Department of Ophthalmology, Sanatorium Hera, Vienna, Austria. Supported by Austrian Science Fund (Fonds zu Förderung der wissenschaftlichen Forschung), Project No Submitted for publication November 29, 2012; revised January 8 and February 13, 2013; accepted February 15, Disclosure: D. Schmidl, None;A. Boltz, None;S. Kaya, None; M. Lasta, None;B. Pemp, None;G. Fuchsjager-Mayrl, None;A. Hommer, None;G. Garhofer, None;L. Schmetterer, None Corresponding author: Leopold Schmetterer, Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria; leopold.schmetterer@meduniwien.ac.at. that the choroid comprises regulatory capacity in vivo and neural mechanisms may have a role in keeping blood flow constant during changes in perfusion pressure. 7 Kiel et al. have shown, in a series of rabbit experiments, that choroidal blood flow remains relatively stable over a wide range of perfusion pressures Studies done in healthy humans also indicated that choroidal blood flow is autoregulated when OPP either was increased during isometric exercise or decreased during an artificial IOP increase In contrast, only few data on optic nerve head (ONH) blood flow regulation during changes in OPP are available. A study performed recently in our laboratory has shown that autoregulation also is present in the ONH, but has a different regulatory range than that observed in the choroid. 11 ONH blood flow (ONHBF) seems to be regulated in response to an acute experimental increase and decrease in OPP. Studies supporting the autoregulatory capacity have been performed in healthy humans, nonhuman primates, cats, and rabbits The mechanisms that mediate this autoregulatory response in the ONH are unclear. Nitric oxide (NO) is a potent vasodilator that is involved in the maintenance of basal vascular tone, in tonic blood pressure regulation, and in the distribution of blood flow. 26 Inhibition of NO synthase (NOS) leads to a decrease in basal retinal and choroidal blood flow in humans and experimental animals This also seems to hold true for the ONH, where inhibition of NOS reduced blood flow in animals and humans. 32,35 In rabbits, NOS inhibition shifted the choroidal pressure flow curve to the right, indicating that NO is involved in choroidal blood flow regulation. 33 Additionally, administration of an unspecific NOS inhibitor attenuated the choroidal response to isometric exercise in healthy humans. 31 The aim of our study was to investigate whether inhibition of NOS influences the response of ONHBF to isometric exercise. A total of 18 healthy subjects received an unspecific NOS inhibitor (N G -monomethyl-l-arginine, L-NMMA), the a- adrenoceptor-agonist phenylephrine, or placebo on three different study days. Since administration of L-NMMA leads to a slight increase in systemic blood pressure at rest, phenylephrine was used as control to overcome the problem that subjects would start at different OPPs. Phenylephrine has been found to have no impact on ocular hemodynamics in the administered dose, while having comparable effects on systemic blood pressure ,39 MATERIALS AND METHODS Subjects Our study was conducted in compliance with the Declaration of Helsinki and the Good Clinical Practice (GCP) guidelines of the European Union. After approval by the Ethics Committee of the Medical University of Vienna, 18 healthy subjects aged between 20 and Investigative Ophthalmology & Visual Science, March 2013, Vol. 54, No Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc.

2 IOVS, March 2013, Vol. 54, No. 3 Optic Nerve Head Blood Flow Regulation years ( years, mean 6 SD) participated in our study. Written informed consent was obtained by all volunteers. Men and women were included in equal parts. The sample size calculation was based on previous unpublished measurements of ONHBF during isometric exercise in our laboratory. A repeated measures ANOVA model was underlying this sample size calculation. These data were used for the sample size calculation selecting an a-error of 0.05 and a power of 0.80, and aimed to measure a difference between treatment arms of 10%. Changes in ONHBF below 10% were assumed to be irrelevant. All subjects had to pass a prestudy screening, which was done during the four weeks before the first study day. This included physical examination and medical history, vital signs, a 12-lead electrocardiogram, determination of height and weight, hematologic status (hemoglobin, hematocrit, red blood cell count, mean corpuscular hemoglobin, white blood cell count, platelet count, activated partial thromboplastin time, thrombin time), clinical chemistry (sodium, potassium, creatinine, alanine aminotransferase, c-glutamyltransferase, total bilirubin, total protein), urine analysis (white blood cell count, nitrite, ph, protein, glucose, ketones, urobilinogen, bilirubin, blood/ hemoglobin), and an ophthalmic examination. If any clinically significant abnormality was found as part of the prestudy screening, including medical history of peripheral vasospasm, the subject was not included. Only subjects with ametropia of less than 3 diopters and anisometropia of less than 1 diopter were allowed to participate in our study. Further exclusion criteria were regular use of medication, regular drug intake during the three weeks before the start of the study (except oral contraceptives), and smoking. Study Design The study was conducted in a randomized, double-masked, placebocontrolled, three-way cross-over design. Subjects were assigned to receive intravenous infusions of either L-NMMA, phenylephrine, or physiologic saline solution as placebo on three different study days. A washout-period of at least four days was scheduled between the study days. On the trial days subjects arrived after a light meal and sleep for 7 to 8 hours. For purposes of ONHBF measurement, the pupil of the eye under study was dilated with tropicamid (Mydriaticum Agepha -Gtt; Agepha, Vienna, Austria). After a resting period of at least 20 minutes, baseline measurements of ocular and systemic hemodynamics were performed. ONHBF was measured continuously for three minutes at baseline. Thereafter, subjects performed squatting for 6 minutes and ONHBF was measured continuously. Squatting was performed in a position where the upper and the lower leg are as close to a right angle as possible. For the subject s security, a nurse was standing behind the subject during the squatting periods. Systemic hemodynamics were assessed every minute. IOP was measured at the end of the squatting period. Thereafter, a resting period of at least 30 minutes was scheduled. When systemic hemodynamics had returned to baseline, study drugs were administered for 20 minutes. During the last 6 minutes of drug administration another squatting period was performed. An illustration of the experimental setup is shown in Figure 1. Drugs and Drug Administration A bolus of 6 mg/kg L-NMMA (Bachem Distribution Services GmbH, Weil am Rhein, Germany) given over 5 minutes was followed by a continuous infusion of 60 lg/kg/min over 15 minutes. Phenylephrine (Neosynephrine; Winthrop Breon Laboratories, New York, NY) was administered at a dose of 1 lg/kg/min over an infusion period of 20 minutes. Physiologic saline solution (as placebo) was infused over a period of 20 minutes. Similar doses of L-NMMA and phenylephrine have been used in previous experiments in our laboratory and were well tolerated To allow for double-masked conditions, phenylephrine and placebo were prepared in two syringes infused subsequently. Methods Noninvasive Measurement of Systemic Hemodynamics. Systolic (SBP), diastolic (DBP), and mean arterial (MAP) pressures were measured on the upper arm by an automated oscillometric device. Heart rate (HR) was recorded concomitantly from a finger pulse oxymetric device (HP-CMS patient monitor; Hewlett Packard, Palo Alto, CA). Laser Doppler Flowmetry (LDF). Measurement of ONHBF was performed by LDF. 40,41 For this purpose, the vascularized tissue was illuminated by coherent laser light. Scattering on moving red blood cells (RBCs) leads to a frequency shift in the scattered light. In contrast, static tissue components do not change light frequency, but lead to randomization of light directions impinging on RBCs. This light diffusing in vascularized tissue leads to a broadening of the spectrum of scattered light, from which mean RBC velocity (vel), the blood volume (vol), and the blood flow (flow) can be calculated in relative units. In our study, laser Doppler flowmetry was performed at the inferior temporal neuroretinal rim to assess ONH blood flow, and care was taken that approximately the same location was used for all subjects. Intraocular Pressure and Ocular Perfusion Pressure. A slitlamp mounted Goldmann applanation tonometer was used to measure IOP. To achieve local anesthesia of the cornea, one drop of oxybuprocaine hydrochloride combined with sodium fluorescein (Thilorbin; Alcon Pharma, Freiburg im Breisgau, Germany) was used before each measurement. OPP was estimated as 2/3 MAP IOP. 4 Data Analysis For analysis of ONHBF, average blood flow data over each minute were used. A repeated measures ANOVA model was used. Differences between the treatments were calculated based on the interaction between time and treatment. Post hoc analyses were done using planned comparisons. For this purpose the time effect was used to characterize the effect of squatting on the outcome parameters. FIGURE 1. Illustration of the experimental setup.

3 1966 Schmidl et al. IOVS, March 2013, Vol. 54, No. 3 TABLE 1. Baseline Values for All Three Study Days (n ¼ 18) In addition, pressure flow relationships were calculated. For this purpose, the data were expressed as percent change in OPP and flow values over baseline. OPP values then were sorted according to ascending values and grouped into 6 groups. As such, 18 values were classified in each group. A statistically significant deviation from baseline flow was defined when the 95% confidence interval (CI) did not overlap with the baseline value anymore. For data description percent changes over baseline were calculated. A P value <0.05 was considered the level of significance. Statistical analysis was done using CSS Statistica for Windows (Version 6.0; Statsoft, Inc., Tulsa, CA). RESULTS Placebo Day Phenylephrine Day L-NMMA Day SBP, mm Hg DBP, mm Hg MAP, mm Hg HR, bpm IOP, mm Hg OPP, mm Hg ONHBF, a.u Data are presented as mean 6 SD. a.u., arbitrary units. No side effects of the administered drugs were observed and all study procedures were well tolerated. None of the subjects had any clinically relevant changes in laboratory parameters at the follow-up examination. All included subjects had an average level of physical fitness and the mean body mass index was kg/m 2. The baseline values for all three study days are presented in Table 1, in which no significant differences were found. Isometric exercise induced a significant increase in MAP during all pretreatment periods (P < 0.01 versus baseline, Fig. 2). Isometric exercise had no effect on IOP during the pretreatment periods (between and mmHg before and between and mm Hg at the end of TABLE 2. Values at Rest before and after Drug Administration on All Three Study Days (n ¼ 18) Pretreatment During Administration Placebo day IOP, mm Hg OPP, mm Hg ONHBF, a.u Phenylephrine day IOP, mm Hg OPP, mm Hg ONHBF, a.u L-NMMA day IOP, mm Hg OPP, mm Hg ONHBF, a.u Data are presented as mean 6 SD. squatting, P ¼ 0.36 between groups). Therefore, changes in OPP during squatting were influenced primarily by changes in MAP. Accordingly, an almost parallel increase in OPP was seen (P < 0.01 versus baseline, Fig. 3). As expected, isometric exercise also caused an increase in HR (Fig. 4). This was highly significant versus baseline (P < 0.01) and seen during all pretreatment periods. The maximum increase in OPP during the pretreatment period was between 55% and 68% on the 3 study days. This increase was paralleled by a maximum increase in ONHBF between 10% and 13% (Fig. 5). The increase in ONHBF was less pronounced than the increase in OPP indicating for some degree of autoregulation. Placebo did not influence baseline systemic or ocular hemodynamic parameters. Administration of phenylephrine led to a significant increase in resting MAP and OPP (P < 0.01 versus placebo each, Fig. 2, Table 2). HR decreased significantly when phenylephrine was administered (P < 0.01 versus placebo, Fig. 4). In contrast, administration of phenylephrine had no impact on baseline IOP or ONHBF under resting FIGURE 2. The effect of isometric exercise on MAP. The first period of squatting was done without drug administration, and the second period during infusion of L-NMMA (circles), phenylephrine (PE, triangles), or placebo (squares). Data are presented as the mean 6 SD (n ¼ 18). *Significant changes versus baseline. #Significant effects of L-NMMA and PE on baseline parameters versus placebo.

4 IOVS, March 2013, Vol. 54, No. 3 Optic Nerve Head Blood Flow Regulation 1967 FIGURE 3. Relative change in OPP during isometric exercise. The first period of squatting was done without drug administration, and the second period during infusion of L-NMMA (circles), PE (triangles), or placebo (squares). Data are presented as the mean 6 SD (n ¼ 18). *Significant changes versus baseline. conditions (P ¼ 0.72 and 0.17 versus placebo, respectively, Table 2). As expected, infusion of L-NMMA induced a significant increase in resting MAP and OPP (P < versus placebo, Fig. 2, Table 2). A decrease in HR compared to that after placebo administration was observed (P ¼ 0.81 versus placebo, Fig. 4). Administration of L-NMMA had no effect on IOP (P ¼ 0.73 versus placebo, Table 2), but a significant decrease in basal ONHBF was observed after administration of L-NMMA ( %). This reduction in ONHBF was significant compared to phenylephrine and placebo (P < 0.01, Table 2). While phenylephrine and L-NMMA significantly increased resting OPP (phenylephrine by %, L-NMMA by % versus baseline, Table 2) no difference in the behavior of the time course of OPP during the squatting periods was observed (P ¼ 0.64 between groups, Fig. 3). In addition there was no difference in the response of ONHBF to isometric exercise between the three groups (P ¼ 0.37, Fig. 5). Pressure flow relationships for all three groups are presented in Figure 6. Neither administration of L-NMMA nor administration of phenylephrine had any impact on the pressure flow relationship. The graphs for all three groups demonstrate the wide autoregulatory range of the ONH during isometric exercise. During the pretreatment squatting periods, ONHBF started to increase consistently at OPP values between 55% and 70% over baseline. During the squatting periods with FIGURE 4. The effect of isometric exercise on heart rate. The first period of squatting was done without drug administration, and the second period during infusion of L-NMMA (circles), PE (triangles), or placebo (squares). Data are presented as the mean 6 SD (n ¼ 18). *Significant changes versus baseline. #Significant effects of PE on baseline parameters versus placebo.

5 1968 Schmidl et al. IOVS, March 2013, Vol. 54, No. 3 FIGURE 5. Relative change in ONHBF during isometric exercise. The first period of squatting was done without drug administration, and the second period during infusion of L-NMMA (circles), PE (triangles), or placebo (squares). Data are presented as the mean 6 SD (n ¼ 18). simultaneous drug administration, ONHBF started to increase consistently at OPP values between 55% and 76% over baseline. DISCUSSION Our data confirmed previous results that the ONH shows autoregulatory capacity during an experimental increase in OPP by isometric exercise. 11,22 As shown in Figure 6, ONHBF started to increase significantly when OPP was raised to values between 55% and 70% over baseline, when no drug or placebo was administered. This is in accordance with data from a previous study performed in our laboratory, where autoregulation in the ONH started to fail at OPP values of 66% over baseline. 11 In contrast, a similar study performed by Movaffaghy et al. found no cutoff point for the upper limit up ONHBF autoregulation, but their study participants only reached OPP values of % over baseline. 22 As in the choroid, NO is an important regulator of basal blood flow in the ONH, since administration of L-NMMA significantly decreased ONHBF. In contrast, administration of phenylephrine, which induced a comparable elevation in basal OPP, did not change ONHBF. L-NMMA increased vascular resistance in the ONH under basal conditions, which is compatible with a study where administration of NO donors decreased vascular resistance in the ONH dose-dependently. 42 Since L-NMMA is an unspecific inhibitor of NOS, we do not know if either endothelial NOS (enos) or neuronal NOS (nnos) is involved in the regulation of baseline ONHBF. As in other vascular beds, we assumed that NO produced via enos in the vascular endothelium has a key role. Studies performed in rats have shown that nnos is expressed in astrocytes in the prelaminar, laminar, and postlaminar region of the ONH, while enos is present throughout the vasculature of the ONH. 43 A postmortem study in healthy eyes found only sparse expression of nnos in the astrocytes throughout the ONH. 44 L-NMMA had no influence on the behavior of ONHBF during isometric exercise. This is in contrast to data obtained in the choroid, where L-NMMA shifted the pressure flow significantly curve to the left. 31 As stated previously, the choroid is richly innervated, while at least the anterior parts of the ONH vasculature seem to lack this innervation. 45 Indeed, the anterior and the postlaminar regions of the ONH have different sources of blood supply. Branches to the anterior region of the ONH derive from the central retinal artery, while the postlaminar region of the ONH and the posterior choroid are supplied by the short posterior ciliary arteries, which are neurally innervated Since an unspecific NOS inhibitor was used in the current as well as in the previous study in FIGURE 6. Pressure flow relationship determined by categorized OPP and ONHBF values during isometric exercise. Relative data were sorted into groups of 18 values, each according to ascending OPP. The dotted line indicates baseline values.

6 IOVS, March 2013, Vol. 54, No. 3 Optic Nerve Head Blood Flow Regulation 1969 which choroidal blood flow was measured, 31 it cannot be ruled out that nnos is responsible for the choroidal response to isometric exercise. 31 It must be kept in mind that LDF is more sensitive to changes in blood flow in the superficial layer in the ONH than to changes in the postlaminar regions. 49 As such, our data cannot necessarily be extrapolated to deeper ONH layers, which may well be regulated by NO during changes in OPP. Our results are supported by a study performed by Takayama et al. in rabbits. 21 In this study, IOP was decreased from either 40 or 60 mm Hg to 10 mm Hg to increase OPP experimentally. During this experiment, an unspecific NOS inhibitor was administered and no difference in the autoregulatory behavior of ONHBF was found compared to placebo. 21 Therefore, for the ONH it is possible that other substances, such as endothelin or prostaglandins, are responsible for the autoregulatory behavior during an experimental increase in OPP. Administration of endothelin-1 significantly decreased ONHBF in healthy humans and endothelin-a receptor blockade modified the response of ONHBF to isometric exercise. 50,51 After topical application of several prostaglandin analogues, a significant increase in ONHBF was found. 52,53 However, the impact of arachidonic acid metabolites on ONHBF during experimental changes in OPP must be investigated further. In conclusion, our data supported previous findings that ONHBF is autoregulated during an experimental increase in OPP. 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