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Physiology and Pharmacology  |   May 2015
Role of Neuronal Nitric Oxide Synthase in Regulating Retinal Blood Flow During Flicker-Induced Hyperemia in Cats
Author Affiliations & Notes
  • Takafumi Yoshioka
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Taiji Nagaoka
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Youngseok Song
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Harumasa Yokota
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Tomofumi Tani
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Akitoshi Yoshida
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Correspondence: Taiji Nagaoka, Department of Ophthalmology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan; nagaoka@asahikawa-med.ac.jp
Investigative Ophthalmology & Visual Science May 2015, Vol.56, 3113-3120. doi:10.1167/iovs.14-15854
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      Takafumi Yoshioka, Taiji Nagaoka, Youngseok Song, Harumasa Yokota, Tomofumi Tani, Akitoshi Yoshida; Role of Neuronal Nitric Oxide Synthase in Regulating Retinal Blood Flow During Flicker-Induced Hyperemia in Cats. Invest. Ophthalmol. Vis. Sci. 2015;56(5):3113-3120. doi: 10.1167/iovs.14-15854.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate how neuronal nitric oxide synthase (nNOS) contributes to regulation of the retinal circulation during rest and flicker stimulation in cats.

Methods.: Using laser Doppler velocimetry, we measured the vessel diameter and blood velocity simultaneously and calculated the retinal blood flow (RBF) in feline first-order retinal arterioles. After intravitreal injections of Nω-Nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, and Nω-propyl-L-arginine (L-NPA), a selective nNOS inhibitor, we continuously monitored the retinal circulation without any perturbations for 2 hours. We then examined the changes in the RBF in response to 16-Hz flicker stimuli for 3 minutes at 2 hours after intravitreal injection of phosphate-buffered saline (PBS) as a control, L-NAME, L-NPA, and thromboxane A2 (TXA2) analogue U46619 as a basal tone-adjusted control.

Results.: After intravitreal injection of L-NAME and L-NPA, the baseline RBF decreased gradually in a dose-dependent manner. In the PBS group, the RBF increased gradually and reached a maximal level after 2 to 3 minutes of flicker stimuli. After 3 minutes of 16-Hz flicker stimuli, the RBF increased by 53.5% ± 3.4% compared with baseline. In the L-NAME and L-NPA groups, the increases in RBF during flicker stimulation were attenuated significantly compared with the PBS group. In the TXA2 group, the reduction in the flicker-induced increase in RBF was comparable to that in the PBS group.

Conclusions.: The current results suggested that increased RBF in response to flicker stimulation may be mediated by nitric oxide (NO) production via nNOS activation.

Retinal vessels dilate and retinal blood flow (RBF) increases as a result of a functional hyperemic response when the retina is stimulated by a flickering light, indicating that retinal neural activity is coupled to blood flow and metabolism.1 Many recent clinical studies have reported that vasodilation of the retinal vessels elicited by flicker stimuli deteriorates in patients with ocular diseases, such as diabetic retinopathy25 and glaucoma.6 However, only a few in vivo animal studies have investigated the molecular mechanism of flicker-induced vasodilation of retinal vessels and functional retinal hyperemia. Kondo et al.7 also reported that intravenous injection of the nonselective nitric oxide synthase (NOS) inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME) prevents a flicker-induced increase in RBF measured using microsphere methods, suggesting that nitric oxide (NO) may play a role in flicker-induced retinal hyperemia. However, the investigators also observed that intravenous injection of L-NAME caused increases in the mean arterial systemic blood pressure (BP) from 110 to 146 mm Hg. In addition, conflicting data have been published regarding the human retina for flicker-induced vasodilation after intravenous injection of nonspecific NOS inhibitor L-NMMA; one human study reported a decreased flicker response in the retinal vessels,8 and another study found an increased response.9 Both human studies showed that the retinal vessel diameter decreased and the systemic BP increased after intravenous injection of L-NMMA. The discrepancy between these previous studies may reflect the effect of increased systemic BP before flicker stimuli. Because we recently reported that acutely elevated systemic BP leads to increased RBF due to release of NO and prostanoids probably through a shear stress-induced vasodilation mechanism in cats,10 it is unclear how NO contributes to flicker-induced retinal hyperemia. 
Moreover, among the three isoforms of the enzyme NOS, that is, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), constitutive NOS (nNOS and eNOS) are thought to continuously synthesize small amounts of NO and contribute to physiological regulation of the ocular hemodynamics.11 Neuronal NOS is present especially in the retina in the ganglion, amacrine, horizontal, and photoreceptor cells and the Müller glial cells,1217 whereas eNOS is expressed mainly in the vascular endothelial cells of the retinal vessels and by the pericytes of the retinal capillaries.1820 We previously confirmed that eNOS contributes to NO production in response to increased shear stress, which is recognized as an index of vascular endothelial function, in response to hypoxic hyperemia21 and acute hypertension.10 In contrast, the role of nNOS in the regulation of the retinal circulation remains unclear. 
In the current study, we examined whether either nNOS or eNOS alone or both are involved in the flicker-induced retinal hyperemia. To prevent increased systemic BP by intravenous injection of L-NAME as mentioned previously, we used intravitreal injections of the nonselective NOS inhibitor L-NAME and the selective nNOS inhibitor Nω-propyl-L-arginine (L-NPA). 
Materials and Methods
Animal Preparation
The Animal Care Committee of Asahikawa Medical University approved the protocols describing the use of cats and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eighty adult cats (2.3–4.5 kg) of either sex were tracheostomized and mechanically ventilated with room air containing 2% sevoflurane. The flow rate of sevoflurane was maintained at 1.5 L/min during the experiment. Catheters were placed in the femoral arteries and vein. The mean arterial BP (MABP) and heart rate were monitored continuously with a transducer (PowerLab; ADInstruments, Inc., Colorado Springs, CO, USA) and recorder (LabChart; ADInstruments, Inc.) in the proximal thoracic descending aorta. Pancuronium bromide (0.1 mg/kg/h) (Daiichi Sankyo Co., Tokyo, Japan) was infused continuously via the femoral vein to maintain skeletal muscle relaxation during the experiment. With the animals prone, the heads were fixed in a stereotaxic instrument. Arterial pH (pH), arterial partial carbon dioxide tension (PaCO2), arterial partial oxygen tension (PaO2), and bicarbonate ion (HCO3) were measured intermittently with a blood gas analyzer (model ABL5; Radiometer, Copenhagen, Denmark). The rectal temperature was measured and maintained between 37°C and 38°C with a heated blanket. The pupils were dilated with 0.5% tropicamide (Santen Pharmaceutical Co., Osaka, Japan). A 0-diopter contact lens (SEED Co. Ltd., Tokyo, Japan) was placed on the cornea, which was protected by instillation of a drop of sodium hyaluronate (Healon; Abbott Medical Optics, Inc., Abbott Park, IL, USA). A 26-gauge butterfly needle was inserted into the anterior chamber and connected to a pressure transducer and a balanced salt solution (Alcon, Fort Worth, TX, USA) reservoir for monitoring and maintaining the intraocular pressure (IOP) at a constant 10 mm Hg, respectively. 
RBF Measurements
A laser Doppler velocimetry system (Laser Blood Flowmeter, model 100; Canon, Inc., Tokyo, Japan) customized for feline use measured the retinal arteriolar diameter (D) (in micrometers) and velocity (V) (mm/s) as described previously.21,22 The RBF in the arterioles (μL/min) was calculated based on the acquired V and D. Laser Doppler measurements of the temporal retinal arterioles were performed in one eye of each animal. The first-order arterioles were chosen for study because they have relatively straight segments and were sufficiently distant from the adjacent vessels for consistent measurements. 
The RBF was calculated using the formula RBF = S × Vmean, where S is the cross-sectional area of the retinal arteriole at the laser Doppler measurement site, assuming a circular cross section, and Vmean is the mean blood V calculated as Vmean = Vmax/2.23 The MABP was determined using the formula MABP = diastolic BP + (systolic BP − diastolic BP)/3, which is the index of systemic BP. Because the cats were prone during the experiments, the ocular perfusion pressure (OPP) was calculated as OPP = MABP – IOP.10,24 The retinal arterial vascular resistance (RVR) was determined by the formula RVR = OPP/RBF.25 The wall shear rate (WSR), an indicator of wall shear stress, was calculated as WSR = 8 × Vmean/D,26 assuming a parabolic flow profile in the arterioles.21 
Flicker Stimulation
We created a light stimulation device (ASA-01; MAYO Corporation, Aichi, Japan) in which 24 white light-emitting diodes (LEDs) (NS6W183BT; Nichia Corporation, Tokushima, Japan) were arranged around the circumference of the laser Doppler velocimetry (LDV) observation lens. The LEDs enabled control of the light volume and frequency (2–64 Hz). The luminance of the stimulus was measured at the retinal plane of a model eye using a Digital Light Meter LX-1053 (Teston Corporation, Yamanashi, Japan). The retinal illuminance was 3750 lux, stimulus duration was 5 ms, and modulation depth was 100%. Ambient light was reduced to 1 lux or less, and the eyes were dark adapted for 2 hours before presentation of flicker stimuli.27 Fundus illumination was used only for alignment before dark adaptation started. 
Intravitreal Injections and Chemicals
A 30-gauge needle (100-μL syringe; Hamilton, Reno, NV, USA) was used to perform the intravitreal injections (3 mm posterior to the limbus) with care taken to not injure the lens and retina.21 The head of the needle was positioned over the optic disc region. L-NAME, L-arginine, D-arginine, bradykinin (BK), sodium nitroprusside (SNP), and U46619 (thromboxane A2 analogue [TXA2]) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). L-NPA was obtained from Cayman Chemicals Co. (Ann Arbor, MI, USA). All drugs were dissolved in phosphate-buffered saline (PBS). The volume of the intravitreal injections was 50 μL, which we confirmed does not alter any retinal circulatory parameters and minimizes the systemic effects of the inhibitors.21 
Changes in RBF to Flicker Stimulation
The measurements of D and V were started 5 minutes before flicker stimulation. The mean of five measurements at 1-minute intervals was recorded as the baseline value. The retina then was stimulated by the flickering light, and the RBF measurements were performed every 30 seconds during the stimulation period. The changes in retinal circulatory parameters were expressed as the percent change from baseline. In the current study, because the blood flow reaches a plateau 2 minutes after flicker stimulation, we expressed the average value of three points at 120 to 180 seconds as the maximal change. 
Effects of NOS Inhibitors
Baseline RBF.
To examine the dose-dependent effects of the NOS inhibitors, L-NPA and L-NAME, on RBF during rest, they were injected at concentrations of 50, 500, 5, and 50 mM. Given that the volume of the feline vitreous cavity is approximately 2.5 mL, 50 μL L-NPA or L-NAME dissolved in PBS was injected into the vitreous for extracellular concentrations of 1, 10, and 100 μM and 1 mM, respectively, near the retinal vessels. Hereafter, we refer to drug concentrations as injected concentrations. We evaluated the changes from baseline by measuring the RBF every 10 minutes for 2 hours after the injection. 
To confirm that the effect of L-NPA was mediated by stereospecific inhibition of the L-arginine/NO pathway, we injected D-arginine (100 mM) or L-arginine (100 mM) simultaneously with the intravitreal L-NPA injection (50 mM). 
BK-Induced Increase in RBF.
Because we confirmed previously that BK causes endothelium-dependent, NO-mediated vasodilation in isolated porcine retinal arterioles,28 we injected BK into the vitreous to induce the endothelium-dependent vasodilation as previously described.29 We also injected SNP as the endothelium-independent vasodilator. In our previous study, the increase in RBF induced by intravitreal injections of BK (50 μM) and SNP (5 mM) reached the maximal level at 120 minutes and persisted for at least 3 hours.29 These concentrations were sufficient for the maximal vasodilatory concentrations of BK and SNP, based on our previous in vitro study.28 To evaluate the roles of eNOS, nNOS, or both in the changes in RBF in response to intravitreal administration of BK, RBF measurements were performed before and 2 hours after intravitreal injection of BK with pretreatment with L-NAME (500 μM), L-NPA (5 mM), and PBS (control). We also performed the same protocol using the NO donor SNP instead of BK in the presence of L-NPA. 
Flicker-Induced Increase in RBF.
To determine if the specific nNOS inhibitor L-NPA blocks flicker-induced hyperemia in the retinal arterioles, we evaluated the changes in RBF in response to flicker stimuli of 16 Hz for 3 minutes before and 2 hours after intravitreal injection of NOS inhibitors or PBS as a control. The intravitreal injections of L-NAME and L-NPA decreased the RBF, which may have some effect on the percent change in RBF during the flicker stimuli. Therefore, we tried to produce a similar reduction in basal RBF to prevent overestimation of the flicker-induced increase in RBF. In the preliminary study, we found that 50 μM TXA2 caused the same reduction of the baseline RBF as L-NAME (500 μM) and L-NPA (5 mM). Therefore, we intravitreously administered TXA2 (50 μM) as a basal tone-adjusted control to examine whether decreased basal blood flow affects the flicker-induced hyperemia in the retinal arterioles. 
Statistical Analysis
All data are expressed as the mean percentage ± standard error of the mean (SEM). Vasoconstrictor responses to L-NPA and L-NAME were calculated as the percentage decrease in RBF. Vasodilatory responses were calculated as the percentage increases of RBF from baseline. For statistical analysis, we used analysis of variance (ANOVA) for repeated measurements, followed by a post hoc comparison using the Dunnett procedure. Group comparisons were performed using the unpaired Student's t-test. P < 0.05 was considered statistically significant. 
Results
Changes in Retinal Circulation in Response to Flicker Stimuli
The maximal changes in RBF in response to different frequencies of flickering light are shown in Figure 1. Based on the results, we selected 16 Hz for the experiments performed later in our in vivo cat model. The time courses of the retinal parameters such as D, V, RBF, WSR, and RVR during 16-Hz flicker stimulation were measured (Table; Fig. 2). After exposure to flicker stimuli, the RBF gradually increased and reached the maximal level after 2 to 3 minutes. After 3 minutes of 16-Hz flicker stimuli, the D, V, and RBF increased by 7.6% ± 0.9%, 32.9% ± 3.2%, and 53.5% ± 3.4%, respectively, compared with baseline. There were no significant changes in any systemic parameters (pH, PaCO2, PaO2, HCO3, MABP, or HR) before, during, and after 16-Hz flicker stimulation (Table). 
Figure 1
 
Effect of different frequencies of flicker stimulation on RBF for the relative changes from 2 to 64 Hz (respectively, n = 6). The stimulation time was 3 minutes and the dark adaptation time was 2 hours. Data are expressed as the mean percentage ± SEM of the basal RBF level.
Figure 1
 
Effect of different frequencies of flicker stimulation on RBF for the relative changes from 2 to 64 Hz (respectively, n = 6). The stimulation time was 3 minutes and the dark adaptation time was 2 hours. Data are expressed as the mean percentage ± SEM of the basal RBF level.
Table
 
Effect of 16-Hz Flicker Stimulation on Retinal Circulation and Systemic Parameters
Table
 
Effect of 16-Hz Flicker Stimulation on Retinal Circulation and Systemic Parameters
Figure 2
 
Time course changes in RBF in response to flicker stimulation as a control (n = 12). The black line represents the period of flicker (3 minutes). The frequency was 16 Hz, modulation depth was 100%, and dark adaptation time was 2 hours. Data are expressed as the mean percentage ± SEM of the baseline. *P < 0.05 compared with baseline values by repeated-measures ANOVA followed by the Dunnett procedure.
Figure 2
 
Time course changes in RBF in response to flicker stimulation as a control (n = 12). The black line represents the period of flicker (3 minutes). The frequency was 16 Hz, modulation depth was 100%, and dark adaptation time was 2 hours. Data are expressed as the mean percentage ± SEM of the baseline. *P < 0.05 compared with baseline values by repeated-measures ANOVA followed by the Dunnett procedure.
Effect of NOS Inhibitors on RBF During Rest
Intravitreal injections with various concentrations of L-NAME (50 and 500 μM and 5 and 50 mM) and L-NPA (50 and 500 μM and 5 and 50 mM) reduced the RBF during rest in a dose-dependent manner 120 minutes after the injections. The RBF decreased by 30.8% ± 2.0% and 26.6% ± 3.3% of baseline, respectively, with 50 mM of L-NAME (Fig. 3A) and L-NPA (Fig. 3B). 
Figure 3
 
The concentration-dependent effect of the intravitreal injection of L-NAME and L-NPA on RBF during rest. We measured the changes from baseline before and 2 hours after intravitreal injection of L-NAME (A) (respectively, n = 5) and L-NPA (B) (respectively, n = 5). The drug concentration indicates the administered concentration. Data are expressed as the mean percentage ± SEM of the preinjection levels. *Significant (P < 0.05) difference from baseline.
Figure 3
 
The concentration-dependent effect of the intravitreal injection of L-NAME and L-NPA on RBF during rest. We measured the changes from baseline before and 2 hours after intravitreal injection of L-NAME (A) (respectively, n = 5) and L-NPA (B) (respectively, n = 5). The drug concentration indicates the administered concentration. Data are expressed as the mean percentage ± SEM of the preinjection levels. *Significant (P < 0.05) difference from baseline.
Effect of L-Arginine and D-Arginine on Responses to L-NPA
The reduction in RBF induced by L-NPA during rest was abolished by coinjection of the NOS substrate L-arginine, whereas coinjection of D-arginine with L-NPA did not affect the L-NPA response (Fig. 4), indicating that the effects of L-NPA were mediated by stereospecific inhibition of the L-arginine/NO pathway. Intravitreal injection of neither L-arginine nor D-arginine caused changes in the RBF (data not shown) during rest or flicker-induced hyperemia in the retinal arterioles. 
Figure 4
 
Effects of L-arginine (L-arg) and D-arginine (D-arg) on RBF in response to the intravitreal injection of L-NPA. The reduction in basal RBF induced by L-NPA (50 mM) is inhibited by L-arginine coinjection (100 mM) but not by D-arginine (100 mM) (respectively, n = 5). Data are expressed as the mean percentage ± SEM of the preinjection levels. *Significant (P < 0.05) difference from L-NPA alone.
Figure 4
 
Effects of L-arginine (L-arg) and D-arginine (D-arg) on RBF in response to the intravitreal injection of L-NPA. The reduction in basal RBF induced by L-NPA (50 mM) is inhibited by L-arginine coinjection (100 mM) but not by D-arginine (100 mM) (respectively, n = 5). Data are expressed as the mean percentage ± SEM of the preinjection levels. *Significant (P < 0.05) difference from L-NPA alone.
Effect of NOS Inhibitors on Vasodilatory Responses to BK
Pretreatment with L-NAME (500 μM) significantly reduced the increase in RBF induced by intravitreal injection of BK (Fig. 5A). In contrast, pretreatment with L-NPA (5 mM) had no significant effect on the increase in RBF after BK (Fig. 5A). The increase in RBF in response to intravitreal injection of SNP was unaffected by pretreatment with L-NPA (Fig. 5B). 
Figure 5
 
Effects of L-NAME and L-NPA on RBF in response to the intravitreal injection of BK (50 μM) and SNP (5 mM). (A) Pretreatment with L-NAME (500 μM) significantly inhibits the BK-induced increase in RBF, but L-NPA (5 mM) has no effect (n = 5 for both) on the BK-induced increase in RBF. *Significant (P > 0.05) differences from BK only. (B) Pretreatment with L-NPA (5 mM) has no effect on the SNP-induced increase in RBF (n = 5 for both). Data are expressed as the mean percentage ± SEM of the preinjection levels 120 minutes after the injection.
Figure 5
 
Effects of L-NAME and L-NPA on RBF in response to the intravitreal injection of BK (50 μM) and SNP (5 mM). (A) Pretreatment with L-NAME (500 μM) significantly inhibits the BK-induced increase in RBF, but L-NPA (5 mM) has no effect (n = 5 for both) on the BK-induced increase in RBF. *Significant (P > 0.05) differences from BK only. (B) Pretreatment with L-NPA (5 mM) has no effect on the SNP-induced increase in RBF (n = 5 for both). Data are expressed as the mean percentage ± SEM of the preinjection levels 120 minutes after the injection.
Effect of Intravitreal Injections of NOS Inhibitors on RBF in Response to Flicker Stimulation
In the PBS group, the RBF did not change significantly 120 minutes after injection, and the RBF increased by 54.7% ± 2.8% in response to flicker stimuli. In the L-NAME and L-NPA groups, the flicker-induced increases in RBF were restricted significantly to 14.8% ± 3.6% and 19.7% ± 3.0% of the baseline compared with the 54.7% increase in the PBS group (Fig. 6). In the TXA2 group, that is, the basal tone-adjusted control, the RBF decreased significantly by 21.8% ± 6.9% of the preinjected baseline 120 minutes after injection, which was comparable to values in the L-NAME and L-NPA groups but did not affect the flicker-induced increase in RBF (49.1% ± 4.7% of baseline), which was comparable to that in the PBS group. 
Figure 6
 
Effects of L-NAME, L-NPA, and TXA2 on RBF during rest (A) and in response to flicker stimulation (B). (A) L-NAME (500 μM, n = 5), L-NPA (5 mM, n = 5), and TXA2 (50 μM, n = 5) reduce basal RBF to a similar extent 120 minutes after the injection without any perturbations. Data are expressed as the mean percentage ± SEM of the preinjection levels. (B) L-NAME (n = 5) and L-NPA (n = 5) significantly attenuate the increased RBF response to flicker stimulation, but TXA2 (n = 5) has no effect (n = 5). Data are expressed as the mean percentage ± SEM of the preflicker stimulation levels. *Significant (P > 0.05) differences from TXA2. N.S., not significant.
Figure 6
 
Effects of L-NAME, L-NPA, and TXA2 on RBF during rest (A) and in response to flicker stimulation (B). (A) L-NAME (500 μM, n = 5), L-NPA (5 mM, n = 5), and TXA2 (50 μM, n = 5) reduce basal RBF to a similar extent 120 minutes after the injection without any perturbations. Data are expressed as the mean percentage ± SEM of the preinjection levels. (B) L-NAME (n = 5) and L-NPA (n = 5) significantly attenuate the increased RBF response to flicker stimulation, but TXA2 (n = 5) has no effect (n = 5). Data are expressed as the mean percentage ± SEM of the preflicker stimulation levels. *Significant (P > 0.05) differences from TXA2. N.S., not significant.
Effect of Intravitreal Injections on Systemic Circulatory Parameters
There were no significant changes in any systemic circulatory parameters (pH, PaCO2, PaO2, HCO3, MABP, or HR) before and 120 minutes after the intravitreal injection of any drugs (Supplemental Tables S1–S4). 
Discussion
Retinal blood flow has been reported to increase by 30% to 60% of the baseline value during flicker stimulation in rats,30 cats,7 baboons,31 and humans.6 The current findings showed a 53.5% increase in RBF with the increases in vessel diameter (7.6%) and blood velocity (32.9%) in anesthetized cats, which agreed with the previously mentioned published studies. The changes in blood velocity especially were much greater than that in the vessel diameter in the previous animal and human studies, suggesting that measurement of RBF calculated by both vessel diameter and blood velocity is necessary to evaluate retinal neurovascular coupling. 
In most previous clinical studies,25 using a computer-based method to analyze fundus photographs, the mean vessel diameters of the retinal arterioles and venule vessel segments were measured for only 20 seconds in response to flicker stimuli at a frequency of 12.5 Hz using a Dynamic Vessel Analyzer (DVA) system (IMEDOS Systems UG, Jena, Germany), which can evaluate only vessel diameter and not blood velocity and blood flow. Riva et al.32 reported that the biphasic response of the optic nerve head blood flow, not RBF, was observed with a rapid increase in the first 15 to 30 seconds, followed by a smaller but sustained slower increase over the next 1 to 2 minutes, suggesting that flicker-induced hyperemia can be completed in 2 to 3 minutes. In the current study, the RBF gradually increased and reached the maximal level (50% of baseline) for 2 to 3 minutes (Fig. 2), which was comparable to the previous finding. Riva et al.32 also reported that the changes in frequencies may affect flicker-induced hyperemia in the optic nerve head blood flow. We also found that 16 Hz caused the maximal increase in RBF in anesthetized cats (Fig. 1). Further clinical study is needed to evaluate whether a longer duration of flicker stimuli produces a more powerful increase in RBF in response to flicker stimuli in humans compared with the previous clinical study. 
In a recent rat study,33 the investigators induced only 15 seconds of flicker stimuli with 5 Hz, which differed from the current study (3 minutes, 16 Hz). Those authors also found that prolonged flicker stimulation evoked increased capillary blood flow continually for the duration of the stimulus (25% increase of the baseline) in the intermediate layer capillaries after rapid dilation of the arterioles. It is likely that the large, slowly developing dilation of the intermediate layer capillaries, which differed markedly from the initial responses of the upstream arterioles, may have been caused by the delayed, marked dilation of the retinal arterioles due to the flow-induced vasodilation in response to the increased wall shear stress in the retinal arterioles. Although we did not measure the rapid response of the retinal arterioles because of the temporal resolution of the LDV system, we believe that longer flicker stimulation would be suitable to evaluate retinal neurovascular coupling. 
Using a computer-based method to analyze fundus photographs, Nguyen et al.2 reported that increases in the retinal arteriolar and venular diameters in response to flicker stimuli were, respectively, 1.4% ± 2.1% and 2.8% ± 2.0% in patients with diabetes and 3.5% ± 2.4% and 4.0% ± 1.8% in healthy subjects. In addition, Garhöfer et al.6 found that flicker-induced vasodilation of the retinal venules (64 seconds, 8 Hz) also decreased in patients with glaucoma (1.8% ± 3.8%) compared with healthy volunteers (2.4% ± 2.6%). These clinical studies were limited in that the change in vessel diameter detected by the DVA system did not give an accurate indication of blood flow or reflect the total changes in the microcirculation. In the current study, we observed an increase in RBF of 53.5% ± 3.4% compared with baseline in response to 16-Hz flicker stimuli, whereas the vessel diameter and blood velocity increased by 7.6% ± 0.9% and 32.9% ± 3.2% compared with baseline, suggesting that measuring the RBF may be more suitable for detecting changes in the retinal microcirculation in a clinical setting compared with a technique to measure only the vessel diameter. Further clinical study is warranted to examine whether the technique to measure RBF may be more useful for studies in diabetic and glaucomatous eyes. 
Based on previous clinical studies, flicker-induced vasodilation clearly decreased in patients with diabetes.2 However, interpretation of the impaired flicker response in diabetes remains controversial. Some researchers have speculated that flicker-induced vasodilation is used as a test to estimate the capability of the endothelial cells of the retinal vessels to release NO, which can evaluate the endothelial function.34 In the current study, we showed for the first time that NO derived from nNOS, not eNOS, plays an important role in functional hyperemia in RBF in response to flicker stimuli in cats, suggesting that the change in RBF in response to flicker stimuli depends mainly on neuronal tissue and not endothelial cells in retinal vessels. In support of our hypothesis, Pemp et al.34 reported only a weak correlation between brachial flow-mediated vasodilation, which is recognized as the current standard for assessing endothelium-dependent vasodilation, and a flicker response in patients with diabetes, hypertension, and hyperlipidemia. Taken together, it is reasonable that NO derived from nNOS in the retinal neuronal tissues primarily may cause flicker-induced vasodilation or hyperemia in RBF. 
However, our findings did not exclude the possible role of eNOS in flicker-induced vasodilation. In the current study, we observed an increased WSR in response to flicker stimuli (Fig. 2), because the increased shear rate represents increased shear stress, which may activate eNOS in the retinal endothelial cells. We reported previously that a flow-induced mechanism is involved during functional hyperemia. Indeed, we confirmed that the increased RBF in response to both systemic hypoxia21 and acute systemic hypertension10 was associated with flow-induced vasodilation via NO production in response to the increased shear rate in cats. Moreover, Hein et al.35 reported that isolated human retinal arterioles dilated in response to increased flow in an NO-dependent manner, indicating that flow-induced vasodilation occurred via NO production from the endothelium of retinal arterioles. In fact, the current study showed that the dilation was only 3.6% ± 1.5% of the baseline at 30 seconds, but further dilation was observed at 2 to 3 minutes of flicker stimulation (a 7.6% increase over baseline), probably due to the flow-induced vasodilation. Taken together, we could not exclude the possible role of eNOS in the flicker-induced hyperemia via the flow-induced mechanism. 
We investigated the possible role of nNOS-derived NO in the flicker-induced increase in RBF in cats using intravitreal injection of L-NPA, which was reported to be a specific nNOS inhibitor with 149-fold and 3000-fold selectivities over eNOS and iNOS, respectively.36 We first found that intravitreal injection of L-NPA caused a significant reduction in RBF during rest in a dose-dependent manner (Fig. 3), indicating that nNOS may play some roles in RBF regulation during rest. In the retinal arteries, the presence of perivascular nitrergic nerves has been reported.37 Therefore, NO derived from the nitrergic nerves also may contribute to regulation of the retinal circulation during rest. 
We also examined the effects of L-NPA on BK-induced increases in RBF to exclude the possibility that L-NPA inhibits eNOS. As expected, our data showed that pretreatment with L-NPA had no significant effect on the vasodilatory response to BK, whereas L-NAME markedly reduced the increase in RBF induced by BK (Fig. 5). Because we confirmed previously that BK can cause endothelium-dependent, NO-mediated vasodilation of isolated porcine retinal arterioles28 and an in vivo increase in RBF in cats,29 the current findings suggested that the L-NPA concentration did not affect the eNOS-mediated effects in the retina in our in vivo cat model. Moreover, the vasodilatory response to the NO donor SNP was unaffected by L-NPA as expected and suggested that vascular smooth muscle was not inhibited by intravitreal injection of L-NPA. 
Previous studies have suggested that NO production is involved in the vasodilatory responses to flicker stimulation in cats, based on inhibition of the flow response by intravenous infusion of L-NAME with increased systemic BP.38 We found previously that acutely elevated systemic BP leads to increased RBF due to release of NO and prostanoids probably through a shear stress-induced vasodilatory mechanism in anesthetized cats, suggesting that increased systemic BP modulates the role of NO in regulation of RBF.10 In the current study, we found that intravitreal injection of L-NAME significantly blunted the increase in RBF in response to flicker stimuli in cats with no change in systemic circulation (Fig. 6B), suggesting that NO produced in the retina plays an important role in flicker-induced retinal hyperemia. More importantly, the L-NPA doses did not affect the BK-mediated vasodilation, and decreased basal flow inhibited the response to flicker stimulation, whereas a basal tone-adjusted control vasoconstrictor (TXA2 analogue U46619) that produced a similar reduction in basal flow did not alter the flicker-induced vasodilation (Fig. 6B). Therefore, these results suggested that local nNOS-derived NO plays an important role in the vasodilatory response to flicker stimulation without affecting basal tone before starting flicker stimulation. Although the precise sites of nNOS-derived NO generation cannot be ascertained from the current results, nNOS protein was expressed in the ganglion, amacrine, horizontal, and photoreceptor cells and the Müller glial cells in the mammalian retina.1217 Further investigation is warranted to examine which cells are responsible for RBF regulation via NO production by nNOS during rest and in response to flicker stimuli. 
The current study had some limitations. First, although our findings suggested that NO may mediate flicker-induced vasodilation, recent studies have suggested that oxygen may modulate the enzymatic activity involved in the vasodilatory response to flicker stimuli in ex vivo rat retina39 and the human retina.40 Further study is needed to elucidate whether oxygen modulates the enzymatic activity involved in the flicker-induced increase in RBF in our in vivo cat model. Second, general anesthesia using sevoflurane and pancuronium bromide may have some effect on the flicker-induced increase in RBF. Although the current data did not provide a definitive explanation for the effect of general anesthesia, we found in our preliminary study that sevoflurane per se did not change the vessel diameter of isolated porcine retinal arterioles (data not shown), and the change in the concentration of pancuronium bromide did not change RBF in cats anesthetized with sevoflurane (data not shown). Third, it is well known that NO formed by iNOS expressed under influences of inflammatory mediators evokes neurodegeneration and cell apoptosis, leading to serious ocular diseases including diabetic retinopathy.11 In our preliminary study, we found that intravitreal administration of a specific iNOS inhibitor, 1400W (Cayman Chemicals Co.), did not affect the baseline RBF and flicker-induced increase in RBF in healthy cats (data not shown), suggesting that iNOS may have little effect on the flicker response in the retina, at least under physiologic conditions. However, we may have to pay attention to the role of iNOS in further clinical studies that targeted diabetic retinopathy because inhibiting iNOS reversed the flicker-induced arteriole dilations in diabetic rat retinas in vivo.39 
In conclusion, the current results suggest that increased RBF in response to flicker stimulation may be mediated by NO production via activation of nNOS. In addition to previous clinical studies that reported close associations between flicker-induced retinal vasodilation and diabetic retinopathy, a recent clinical study reported a significant correlation between electroretinography and the flicker response measured by the DVA system in patients with diabetes,41 suggesting that the impaired flicker response in diabetes also reflects inner retinal neuronal dysfunction. Taken together, evaluation of the changes in RBF in response to flicker stimulation may shed light on our understanding of the pathogenesis of diabetic retinopathy, which has two components, that is, vascular and neurodegenerative disorders. 
Acknowledgments
Supported by Grant-in-Aid for Scientific Research (B) 25293352 and Challenging Exploratory Research 25670724 from the Ministry of Education, Science, and Culture, Tokyo, Japan (TN). The authors alone are responsible for the content and writing of the paper. 
Disclosure: T. Yoshioka, None; T. Nagaoka, None; Y. Song, None; H. Yokota, None; T. Tani, None; A. Yoshida, None 
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Figure 1
 
Effect of different frequencies of flicker stimulation on RBF for the relative changes from 2 to 64 Hz (respectively, n = 6). The stimulation time was 3 minutes and the dark adaptation time was 2 hours. Data are expressed as the mean percentage ± SEM of the basal RBF level.
Figure 1
 
Effect of different frequencies of flicker stimulation on RBF for the relative changes from 2 to 64 Hz (respectively, n = 6). The stimulation time was 3 minutes and the dark adaptation time was 2 hours. Data are expressed as the mean percentage ± SEM of the basal RBF level.
Figure 2
 
Time course changes in RBF in response to flicker stimulation as a control (n = 12). The black line represents the period of flicker (3 minutes). The frequency was 16 Hz, modulation depth was 100%, and dark adaptation time was 2 hours. Data are expressed as the mean percentage ± SEM of the baseline. *P < 0.05 compared with baseline values by repeated-measures ANOVA followed by the Dunnett procedure.
Figure 2
 
Time course changes in RBF in response to flicker stimulation as a control (n = 12). The black line represents the period of flicker (3 minutes). The frequency was 16 Hz, modulation depth was 100%, and dark adaptation time was 2 hours. Data are expressed as the mean percentage ± SEM of the baseline. *P < 0.05 compared with baseline values by repeated-measures ANOVA followed by the Dunnett procedure.
Figure 3
 
The concentration-dependent effect of the intravitreal injection of L-NAME and L-NPA on RBF during rest. We measured the changes from baseline before and 2 hours after intravitreal injection of L-NAME (A) (respectively, n = 5) and L-NPA (B) (respectively, n = 5). The drug concentration indicates the administered concentration. Data are expressed as the mean percentage ± SEM of the preinjection levels. *Significant (P < 0.05) difference from baseline.
Figure 3
 
The concentration-dependent effect of the intravitreal injection of L-NAME and L-NPA on RBF during rest. We measured the changes from baseline before and 2 hours after intravitreal injection of L-NAME (A) (respectively, n = 5) and L-NPA (B) (respectively, n = 5). The drug concentration indicates the administered concentration. Data are expressed as the mean percentage ± SEM of the preinjection levels. *Significant (P < 0.05) difference from baseline.
Figure 4
 
Effects of L-arginine (L-arg) and D-arginine (D-arg) on RBF in response to the intravitreal injection of L-NPA. The reduction in basal RBF induced by L-NPA (50 mM) is inhibited by L-arginine coinjection (100 mM) but not by D-arginine (100 mM) (respectively, n = 5). Data are expressed as the mean percentage ± SEM of the preinjection levels. *Significant (P < 0.05) difference from L-NPA alone.
Figure 4
 
Effects of L-arginine (L-arg) and D-arginine (D-arg) on RBF in response to the intravitreal injection of L-NPA. The reduction in basal RBF induced by L-NPA (50 mM) is inhibited by L-arginine coinjection (100 mM) but not by D-arginine (100 mM) (respectively, n = 5). Data are expressed as the mean percentage ± SEM of the preinjection levels. *Significant (P < 0.05) difference from L-NPA alone.
Figure 5
 
Effects of L-NAME and L-NPA on RBF in response to the intravitreal injection of BK (50 μM) and SNP (5 mM). (A) Pretreatment with L-NAME (500 μM) significantly inhibits the BK-induced increase in RBF, but L-NPA (5 mM) has no effect (n = 5 for both) on the BK-induced increase in RBF. *Significant (P > 0.05) differences from BK only. (B) Pretreatment with L-NPA (5 mM) has no effect on the SNP-induced increase in RBF (n = 5 for both). Data are expressed as the mean percentage ± SEM of the preinjection levels 120 minutes after the injection.
Figure 5
 
Effects of L-NAME and L-NPA on RBF in response to the intravitreal injection of BK (50 μM) and SNP (5 mM). (A) Pretreatment with L-NAME (500 μM) significantly inhibits the BK-induced increase in RBF, but L-NPA (5 mM) has no effect (n = 5 for both) on the BK-induced increase in RBF. *Significant (P > 0.05) differences from BK only. (B) Pretreatment with L-NPA (5 mM) has no effect on the SNP-induced increase in RBF (n = 5 for both). Data are expressed as the mean percentage ± SEM of the preinjection levels 120 minutes after the injection.
Figure 6
 
Effects of L-NAME, L-NPA, and TXA2 on RBF during rest (A) and in response to flicker stimulation (B). (A) L-NAME (500 μM, n = 5), L-NPA (5 mM, n = 5), and TXA2 (50 μM, n = 5) reduce basal RBF to a similar extent 120 minutes after the injection without any perturbations. Data are expressed as the mean percentage ± SEM of the preinjection levels. (B) L-NAME (n = 5) and L-NPA (n = 5) significantly attenuate the increased RBF response to flicker stimulation, but TXA2 (n = 5) has no effect (n = 5). Data are expressed as the mean percentage ± SEM of the preflicker stimulation levels. *Significant (P > 0.05) differences from TXA2. N.S., not significant.
Figure 6
 
Effects of L-NAME, L-NPA, and TXA2 on RBF during rest (A) and in response to flicker stimulation (B). (A) L-NAME (500 μM, n = 5), L-NPA (5 mM, n = 5), and TXA2 (50 μM, n = 5) reduce basal RBF to a similar extent 120 minutes after the injection without any perturbations. Data are expressed as the mean percentage ± SEM of the preinjection levels. (B) L-NAME (n = 5) and L-NPA (n = 5) significantly attenuate the increased RBF response to flicker stimulation, but TXA2 (n = 5) has no effect (n = 5). Data are expressed as the mean percentage ± SEM of the preflicker stimulation levels. *Significant (P > 0.05) differences from TXA2. N.S., not significant.
Table
 
Effect of 16-Hz Flicker Stimulation on Retinal Circulation and Systemic Parameters
Table
 
Effect of 16-Hz Flicker Stimulation on Retinal Circulation and Systemic Parameters
Supplement 1
Supplement 2
Supplement 3
Supplement 4
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