Role of NO in the Control of Choroidal Blood Flow during a Decrease in Ocular Perfusion Pressure

Abstract

purpose. The study was conducted to investigate whether the l-arginine/nitric oxide system plays a role in choroidal blood flow (ChBF) regulation during a decrease in ocular perfusion pressure (OPP).

methods. Experiments were performed on 3 days in a randomized double-masked, placebo-controlled, three-way crossover design. On different study days, subjects received intravenous infusions of N ^G-monomethyl-l-arginine (l-NMMA), phenylephrine, or placebo. Intraocular pressure was raised in stepwise increments using the suction cup method. Choroidal blood flow (ChBF, laser Doppler flowmetry), mean arterial blood pressure (MAP), and IOP were assessed. Ocular perfusion pressure was calculated as OPP = \({2}/{3}\) (MAP − IOP). For correlation analysis all OPP/ChBF data pairs from all subjects were pooled independent of time point of measurement. Then, the pooled data were sorted according to OPP, and correlation analyses were performed.

results. l-NMMA and phenylephrine increased resting OPP by +17% ± 18% and +14% ± 21%, respectively (P < 0.05). l-NMMA reduced resting ChBF by −21% ± 17% (P < 0.05). The relative decrease in OPP during suction cup application was comparable with all drugs administered. The decrease in OPP was paralleled by a significant decrease in ChBF (maximum between −39% and −47%), which was less pronounced, however, than the decrease in OPP (maximum between −69% and −74%). Neither placebo nor l-NMMA, nor phenylephrine, influenced the OPP/ChBF relationship.

conclusions. The data confirm previously published observations that the choroid shows some regulatory capacity during reduced OPP. The l-arginine/nitric oxide-system plays a role in the maintenance of basal vascular tone but seems not to be involved in the choroidal vasodilator response when IOP is increased.

Nitric oxide (NO) is a key candidate for regulating ocular blood flow during changes in perfusion pressure. Obviously, vascular resistance decreases when blood flow is kept constant during a decrease in perfusion pressure and increases during an increase in perfusion pressure. According to the work of Kiel ¹ employing laser Doppler flowmetry (LDF) in the rabbit, NO is a key candidate to control vascular tone during a changes in perfusion pressure. On the other hand, Koss ² failed to detect an effect of an NO synthase inhibitor on the choroidal pressure–flow relationship in the cat during a decrease in perfusion pressure. In humans NO synthase inhibition alters the ocular perfusion pressure (OPP)–choroidal flow relationship during an increase in perfusion pressure induced by isometric exercise. ³ Whether this truly indicates a role for NO in human choroidal autoregulation is unclear, however, because the neural input to the choroid cannot be measured. NOS inhibition also modulated the response of ONH blood flow assessed with hydrogen clearance to an increase in IOP. ⁴  

In the present study we administered the NO synthase (NOS) inhibitor N ^G-monomethyl-l-arginine (l-NMMA), to clarify whether NO plays a role in the decrease in choroidal vascular resistance during a decrease in OPP. A problem that arises during systemic administration of l-NMMA is that it increases systemic blood pressure. Hence, pressure flow relationships at baseline and after pharmacological intervention are difficult to compare, because they start at different OPPs. The present study was conducted to overcome this problem by using the α-receptor agonist phenylephrine as a control substance in a dose assumed to induce comparable effects on blood pressure. 

Materials and Methods

The present study was performed in compliance with the Declaration of Helsinki and the Good Clinical Practice (GCP) guidelines. The study protocol was approved by the Ethics Committee of the Medical University of Vienna. The nature of the study was explained to all subjects, and they gave written consent to participate. A total of 19 subjects were included in the study. All subjects had been drug free for at least 3 weeks before inclusion and passed a prestudy screening during the 4 weeks before the first study day, which included medical history and physical examination, 12-lead electrocardiogram; complete blood count; activated partial thromboplastin time; thrombin time; clinical chemistry; hepatitis A, B, and C and HIV serology; urine analysis; and an ophthalmic examination. Subjects were excluded if any abnormality was found during the screening, unless the investigators considered an abnormality to be clinically irrelevant. In addition, subjects with ametropia of more than 1 D, anisometropia more than 1 D, or any evidence of eye disease that might interfere with the purpose of the present trial were excluded. 

Experimental Design

The study was performed in a randomized, double-masked, placebo-controlled, three-way crossover design. Three trial days were scheduled for each subject, with a washout period of at least 4 days. All volunteers were asked to refrain from alcohol and caffeine for at least 12 hours before the trial day. In addition, subjects were instructed to have 7 to 8 hours of sleep and a light breakfast before arriving at the Department of Clinical Pharmacology on the trial day. An intravenous cannula (Venflon; SP Services, Telford, UK) was inserted into an antecubital vein for drug infusion. After a 20-minute resting in sitting position baseline measurements of ocular and systemic hemodynamics were performed. Choroidal blood flow was measured continuously for 3 minutes at baseline. Thereafter, the suction cup was applied with a suction of 50 mm Hg. The suction was then increased in three consecutive steps to 100, 150, and 200 mm Hg. Each suction level was maintained for 2 minutes during which choroidal blood flow (ChBF) was continuously measured. After completion of these experiments, a 30-minute resting period was scheduled. Thereafter, the procedure with the suction cup was repeated and at each incremental step IOP was measured instead of ChBF. After another 30-minute resting period, drug administration was started for 20 minutes. Incremental increases in IOP were repeated during the last 8 minutes of drug administration. Choroidal blood flow was measured continuously 3 minutes before and during suction cup application. During drug infusion, IOP was measured at baseline and at the end of the experiment. Systemic blood pressure was assessed every minute. Pulse rate and a real-time ECG were monitored continuously. Subjects were monitored throughout the infusion period and until all parameters returned to baseline. 

Study Medication and Measurements

[scap]l-NMMA (Clinalfa, Läufelfingen, Switzerland) was given intravenously as a bolus of 6 mg/kg over 5 minutes followed by a continuous infusion of 60 μg/kg/min over 15 minutes. ^{3 5} Phenylephrine (Neosynephrine; Abbott Laboratories, North Chicago, IL) was administered as intravenous infusion, 1 μg/kg/min over 20 minutes. ^{3 6} Physiologic saline solution was administered intravenously over 20 minutes as the placebo control. 

To ensure double-masked conditions phenylephrine and placebo infusions were prepared in two syringes each and given as a bolus followed by a continuous infusion as required for the l-NMMA dose regimen. 

Systemic Hemodynamics.

Brachial artery blood pressures: systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure were monitored on the upper right arm by an automated oscillometric device. Pulse rate was automatically recorded from a finger pulse oximeter (HP-CMS monitor; Hewlett Packard, Palo Alto, CA). 

Laser Doppler Flowmetry.

Continuous measurements of subfoveal ChBF were performed by LDF, as described in principle previously. ⁷ With this technique, the vascularized tissue is illuminated by coherent laser light. Light scattered by the moving red blood cells undergoes a frequency shift. In contrast, static tissue scatterers do not change the light frequency, but lead to randomization of light directions impinging on red blood cells. Hence, red blood cells receive light from numerous random directions. As the frequency shift is dependent not only on the velocity of the moving red blood cells, but also on the angle between the incident and the scattered light, scattering of the light in tissue broadens the Doppler shift power spectrum. From this spectrum, hemodynamic parameters can be determined based on a theory of light-scattering in tissue. ⁸ In the present study, a compact laser Doppler flowmeter, which had been described in detail previously, was used. ^{9 10} All measurements were performed in the fovea by asking the subject to directly fixate the beam. 

Intraocular Pressure.

A slit lamp mounted Goldmann applanation tonometer was used to measure intraocular pressure. Before each measurement, 1 drop of 0.4% benoxinate hydrochloride combined with 0.25% sodium fluorescein was used for local anesthesia of the cornea. 

Suction Cup Method.

The IOP was raised by applying a method described by Ulrich et al. ¹¹ In the present study, we used an automatic suction pump that is connected by plastic tubing to a rigid standardized 11-mm-diameter plastic suction cup. After local anesthesia was topically applied, the suction cup was placed on the temporal sclera with the anterior edge at least 1 mm from the limbus. The vacuum was increased in increments from 50 to 200 mm Hg to produce targeted IOP increases of approximately +17, +30, +40 and +50 mm Hg above baseline. ¹²  

Data Analysis

Ocular perfusion pressure was calculated as OPP = \({2}/{3}\) (MAP − IOP). ¹³ All blood flow values measured at OPPs below 10 mm Hg were excluded from statistical analysis because of problems in LDF signal analysis at very low flow rates, as discussed in detail by Riva et al. ¹⁴  

For correlation analyses, the values of ChBF were expressed as a percentage change from baseline (Δ%). All other data are presented in absolute values. P < 0.05 was considered significant. Statistical analysis was performed with commercial software (CSS Statistica for Windows; Statsoft Inc., Tulsa, OK). A three-way, repeated-measures ANOVA model was used to analyze the data during l-NMMA or phenylephrine infusion versus placebo infusion with OPP as a covariable. Changes versus baseline during suction application were tested with planned comparisons for post hoc analyses. 

To evaluate the pressure-flow relationship, relative data were sorted according to ascending OPP values. For this purpose the following procedure was used: The relative changes in ChBF induced by suction were calculated. Thereafter, the OPP and relative ChBF data pairs from all subjects were pooled. The data were then sorted according to ascending OPP values and divided into four groups of ΔOPP and ΔChBF data pairs, each comprising an equal number of data pairs. For each group, the mean value, 95% confidence intervals, and SEM were calculated. In addition, a second-order polynomial fit, based on the individual percent change in ChBF and the absolute OPP data, was performed. 

Results

After the screening, 19 healthy male subjects were enrolled. One subject was excluded on the first study day due to elevated IOP at baseline (23 mm Hg). Two subjects were excluded during the first study day because of insufficient target fixation. Two subjects withdrew their consent to participate after the first study day. Fourteen subjects (aged 25 ± 4 years; BMI, 23 ± 2 kg/m²) completed the study according to the protocol. No adverse events were observed except localized mild conjunctival hyperemia. 

Table 1refers to the baseline characteristics of the remaining 14 participants. There were no significant differences between the baseline values on the three trial days. The application of the suction cup at different suction levels caused a highly significant increase in IOP (to average: +18, +26, +34, and +41 mm Hg). This effect was comparable on all study days. Administration of phenylephrine (−0.1 ± 1.9 mm Hg, NS) or placebo (−0.7 ± 1.6 mm Hg, NS) did not affect the IOP. l-NMMA caused a small but significant decrease in IOP before application of the suction cup (−2.4 ± 2.3 mm Hg; P < 0.05 versus baseline). Administration of the placebo did not change the arterial blood pressure. l-NMMA and phenylephrine increased MAP significantly (+8% ± 9%, and +10% ± 13%, respectively, P < 0.01 versus baseline), but there was no significant difference in the systemic hypertensive response to the two drugs. Placebo and phenylephrine had no consistent effect on ChBF at baseline. l-NMMA, however, decreased ChBF significantly before application of suction cup (−21% ± 17%; P = 0.005 versus baseline). 

The calculated changes in OPP are shown in Figure 1 . During the pretreatment periods, we observed a pronounced reduction in OPP when the suction was applied (P < 0.001 versus baseline). The experimentally induced changes in OPP were comparable during the pretreatment periods on all study days (P = 0.63). Placebo administration did not influence OPP changes during the application of suction. Since the administration of l-NMMA or phenylephrine increased arterial blood pressure, the OPP was higher compared with pretreatment (P < 0.05) and placebo (P < 0.05) during the infusion of both drugs (Fig. 1) . The increase in OPP during placebo, l-NMMA, or phenylephrine was not different (P = 0.81). 

The decrease in OPP was paralleled by a significant decrease in ChBF during the pretreatment periods (maximal reduction between −38.8% and −47.4%, P < 0.001, Table 2 ). Similar changes in ChBF were observed during infusion of placebo (−41.3% at maximum, P < 0.001), l-NMMA (−49.4% at maximum, P < 0.001), and phenylephrine (−39.4% at maximum, P < 0.001). However, the reduction of ChBF was less pronounced than the decrease in OPP during all stimulation periods (Table 2) . There was no difference in the ChBF response on the three study days (P = 0.43). 

The OPP/ChBF relationships during the application of suction levels are presented in Figure 2 . During the pretreatment periods, a change of approximately −70% in OPP caused a change of approximately −40% in ChBF only. Neither placebo nor l-NMMA, nor phenylephrine, however, influenced the OPP/ChBF relationships. 

Discussion

The data from the present trial indicate that the l-arginine/NO-system is not involved in the regulatory mechanisms of the choroid vessels during a decreased OPP induced by an experimental IOP increase in humans. The present study, however, confirmed that a reduction in OPP is paralleled by a decrease in ChBF, which is less pronounced than the OPP decrease, as has been shown in various experiments in humans. ^{15 16 17}  

The resting ChBF was significantly reduced after administration of l-NMMA. This result confirms our previous findings, ¹⁸ indicating the important role of l-arginine/NO-system in maintenance of basal ChBF. In a recent study, we have shown that inhibition of NOS significantly alters the regulation of ChBF as OPP increases. ³ During isometric exercise, the upper limit of regulatory capacity in the choroid appears to be approximately 50% to 60% above the baseline OPP. ^{19 20 3} When l-NMMA was administered, the ChBF remained unchanged despite an increase in OPP of more than 70%. In contrast, during infusion of placebo or phenylephrine, ChBF started to increase early, when the OPP was approximately 50% above baseline in this study population. ³ These results suggest that the l-arginine/NO system is also involved in regulatory mechanisms in the choroid during increased OPP in humans. 

The present study investigated the influence of l-NMMA on ChBF during decreased OPP. According to Kiel, ¹ l-NAME (NO synthase inhibitor) caused a reduction of ChBF over a wide perfusion pressure range compared with vehicle in rabbits. Our findings in humans are in partial contradiction of the results of Kiel. ¹ The data of the present study do not indicate an effect of l-NMMA on the OPP/ChBF relationship if OPP is decreased during an IOP increase. This finding may be related to different experimental conditions. Kiel ¹ reduced OPP by manipulations of blood pressure using hydraulic occluders placed around the descending thoracic aorta and the inferior vena cava. This method allows precise stepwise changes of OPP. In humans the most widely used way to decrease OPP without drugs is to use the suction cup method. Since the suction cup does not keep adhesive on the sclera at a suction of less than 50 mm Hg, it is not possible to increase IOP to levels of only a few millimeters of mercury above baseline. In the present study the smallest reduction in OPP during application of 50 mm Hg suction was between −25% and −85% (average, −40%). This finding may be of importance if NO contributes only to ChBF regulation in humans during small reductions in OPP, as previously observed in newborn pigs. ²¹ On the other hand, Koss ² did not observe any influence of l-NAME on regulatory mechanisms in the anterior choroid in cats. The different results of NO-synthase inhibition on ChBF during decreased OPP could also be related to different vascular responses in various species. In addition, studies were performed with the animals under anesthetics, which may have influenced ChBF regulation to an unknown degree. 

The role of the l-arginine/NO system in vascular regulatory mechanisms in the cerebral circulation has been studied extensively since the late 1980s. There is evidence that NO also takes a significant part in the maintenance of resting cerebral blood flow. ^{22 23} The recent findings of Talman and Nitschke Dragon ²⁴ support the hypothesis that NO contributes to parasympathetically mediated cerebral vasodilatation during acute hypertension in rats. In this study, nNOS was selectively inhibited by administering propyl-l-arginine directly adjacent to the observed cerebral vessel. This local nNOS blockade significantly attenuated the changes in pial arterial diameter induced by systemic hypertension. ²⁴ Several studies have been undertaken to investigate the role of the l-arginine/NO system in cerebral autoregulation during hypotension, but the results are inconsistent. Intraperitoneal administration of 50 mg l-NAME twice daily over 4 days did not affect autoregulatory processes in anesthetized rats during reduced arterial blood pressure. ²⁵ In this study, the [¹⁴C]iodoantipyrine autoradiographic method was used for blood flow measurements. Buchanan and Phillis ²⁶ investigated the effects of intravenously administered 30 mg/kg l-NAME on cerebral circulation in rats during hypotension. Using the venous outflow procedure, no changes in the lower limit of autoregulation were observed. Some other studies in animals also suggest that l-arginine/NO-system does not play a significant role in cerebral regulatory mechanisms during hypotension. ^{27 28} Most of the investigators, however, report an increase in the lower limit of autoregulation during the administration of NO-synthase inhibitors. ^{29 30 31 32 33} Administration of l-NNA (N _ω-nitro-l-arginine), a nonselective NOS inhibitor, shifts the autoregulatory curve to the right, as shown with LDF in rats. ^{30 31 32 33} Similar results were obtained during administration of l-NMMA, as measured with the [¹⁴C]iodoantipyrine autoradiographic method in rats ²⁹ and the photoelectric method in cats. ³⁰ The reason for the contradictory results is not completely clear. First, the studies were performed with various drugs and by different methods. Second, systemic administration of NOS inhibitors causes an increase in blood pressure, which complicates interpretation of the obtained data in some studies. Third, it is known that cerebral autoregulation varies in different brain regions. Therefore, measurements of global cerebral circulation incorporate unequal vascular beds. In fact, the investigators who measured blood flow locally observed more significant changes in the lower limit of autoregulation during NOS inhibition ^{30 31 32 33} than did those who assessed global cerebral circulation. ^{26 27} To compare cerebral data with our findings in the choroid is difficult, however, because no human data are available. 

Both sympathetic and parasympathetic nerves have been found in the choroid. ^{34 35 36 37} An increase of blood pressure activates the sympathetic system resulting in vasoconstriction to protect the tissue against overperfusion. ³⁸ The parasympathetic system seems to be less involved. ³⁹ Whether an experimental increase in IOP, as induced using the suction cup method, stimulates the sympathetic or parasympathetic nerves is unknown. The parasympathetic perivascular nerves in the choroid are immunoreactive for nitric oxide synthase, ^{40 41 42} indicating the possibility of nitrergic blood flow control in the choroid. Kiel, ¹ who showed the influence of NO synthase inhibition on the lower autoregulatory limit in rabbits, hypothesized that both neuronal and endothelial NO play a role in choroidal regulatory mechanisms. Whether neural input plays a role in ChBF regulation during an artificial increase in IOP as used in the present study is unclear. 

Important limitations in human studies on this topic include different baseline OPPs as well as the wide interindividual variability of suction cup–induced changes in OPP. We tried to overcome these problems by using relative data versus baseline and by pooling all ChBF–OPP data pairs independently of the time point of measurement. The data were then sorted according to ascending OPP values and divided into four groups. In addition, we could not use all blood flow data obtained because some of them were measured during very low perfusions pressures. If blood flow is below a certain level, LDF analysis as used in the present study does not work appropriately. ¹⁴ Briefly, red blood cells velocities increase during low OPP, because of artifacts in the signal analysis. This effect occurs when a significant portion of the power of the laser Doppler spectrum is below the low-cutoff frequency of the analysis. ¹⁴ We also observed this effect in our study, particularly if the OPP decreased below 10 mm Hg. Therefore, we had to exclude all the affected values from statistical analysis. Consequently, we lost between 9 (during phenylephrine infusion) and 31 (during pretreatment on placebo day) ChBF–OPP data pairs from the statistical analysis. This problem is not a usual for the LDF system (Oculix, Philadelphia, PA), but is due to selection of the low-cut filter used for our experiments to avoid movement-related Doppler artifacts. Another limitation of the present study is that IOP measurements and blood flow measurements during suction were not performed simultaneously. We have shown previously, however, that a 30-minute resting interval is sufficient to obtain reproducible IOP responses during suction (unpublished data, 2007). In the present study, we at least showed that IOP did not change versus baseline before the second suction cup period. 

Limitations in human studies of ChBF using LDF have been discussed in detail elsewhere. ⁴³ Briefly, using this device we were able to perform measurements only in the subfoveal choroid, and the depth of measurements is unknown. Therefore, our results may be not applicable for peripheral parts of the choroid. In addition, with the LDF technique, only red blood cell flux, but not blood flow in its strictest sense, is measured. In case of changes in local hematocrit, the relation between red blood cell flux and blood flow may therefore be altered. However, hematocrit changes during the study days in the present trial are unlikely. 

In conclusion, our data confirm previously published observations that the choroid shows some regulatory capacity during reduced OPP. The l-arginine/nitric oxide system plays a role in the maintenance of basal vascular tone in the choroid, but seems not to be involved in the regulatory mechanisms induced by an increase in IOP. 

 Supported by Austrian Science Fund (Fonds zur Förderung der wissenschaftlichen Forschung), project number P15970.

 Submitted for publication December 17, 2007; revised February 7, May 23, and July 17, 2008; accepted November 3, 2008.

 Disclosure: C. Simader, None; S. Lung, None; G. Weigert, None; J. Kolodjaschna, None; G. Fuchsjäger-Mayrl, None; L. Schmetterer, None; E. Polska, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Baseline Data on Each Study Day

	Placebo Day	l-NMMA Day	Phenylephrine Day
MAP (mm Hg)	76 ± 7	78 ± 5	77 ± 5
PR (beats per minute)	70 ± 10	71 ± 10	71 ± 8
IOP (mm Hg)	13 ± 2	13 ± 2	13 ± 2
OPP (mm Hg)	38 ± 5	39 ± 3	39 ± 4
ChBF (arbitrary units)	45 ± 20	42 ± 11	45 ± 18

 Mean ± SD; n = 14.

Figure 1.

 

The effects of suction cup application on OPP. The first period was performed without drug administration (pretreatment, left). The second period was performed during the administration of placebo (•), l-NMMA (▪) or phenylephrine (▴). Data are presented as the mean ± SEM (n = 14). *Significant changes versus baseline; #significant changes versus placebo.

Table 2.

 

ChBF during the Study Days

	Baseline	Suction Cup (mm Hg)
				50	100	150	200
Placebo Day
Pretreatment
ChBF (%)	100	78.1 ± 12.0*	64.5 ± 9.7*	52.6
n	14	13	10	1	0
During placebo infusion
ChBF (%)	100	76.0 ± 17.4*	62.7 ± 14.5*	58.7 ± 18.9*	66.9
n	14	14	10	5	1
l-NMMA Day
Pretreatment
ChBF (%)	100	83.1 ± 9.4*	72.2 ± 13.3*	62.3 ± 11.4*	59.4
n	14	12	11	5	1
During l-NMMA infusion
ChBF (%)	100	93.5 ± 26.6	71.2 ± 15.5*	50.6 ± 19.4*	55.9 ± 11.5
n	14	14	12	8	3
Phenylephrine Day
Pretreatment
ChBF (%)	100	77.5 ± 10.6*	68.7 ± 15.6*	61.2 ± 21.0
n	14	13	11	4	0
During phenylephrine infusion
ChBF (%)	100	85.3 ± 11.1*	69.8 ± 16.7*	63.5 ± 6.5*	60.6 ± 17.4*
n	14	14	14	11	7

 Data are the percentage change from baseline. Values measured during ocular perfusion pressure below 10 mm Hg were excluded. Data are presented as the mean ± SD. n, number of subjects.

*  Significant changes versus baseline.

Figure 2.

 

Pressure-flow relationship using the categorized OPP–ChBF data during the study days. Relative data of ChBF and absolute data of OPP from all subjects were sorted according to ascending OPPs into three groups of OPP and ChBF data pairs. Open symbols: pretreatment data on different study days; solid symbols: data during infusion of the drugs: placebo, l-NMMA, or phenylephrine. Circles: individual data; triangles: grouped data. Data were fitted by using a second-order polynomial. Dotted lines: pretreatment periods; solid lines: treatment periods. Data are presented as the mean ± SEM.