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(Investigative Ophthalmology and Visual Science. 2002;43:784-789.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Evidence that Nitric Oxide Is Involved in Autoregulation in Optic Nerve Head of Rabbits

Takashi Okuno, Hidehiro Oku, Tetsuya Sugiyama, Ying Yang and Tsunehiko Ikeda

From the Department of Ophthalmology, Osaka Medical College, Osaka, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the role played by nitric oxide (NO) in the autoregulation of circulation in the optic nerve head (ONH).

METHODS. The intraocular pressure (IOP) was increased and maintained at 50 mm Hg by the infusion of balanced saline solution (BSS) into the anterior chamber of albino rabbits. Experiments were performed with or without an intravenous injection of 10, 20, or 50 mg/kg NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. The blood flow in the ONH was evaluated by the hydrogen clearance method, and NO metabolites (nitrite and nitrate) were measured in the ONH under the same experimental conditions in other rabbits. Visual evoked potentials (VEPs) were recorded before the IOP elevation and every 15 minutes during the 60 minutes of elevation. The effect of elevated IOP on the VEPs and the hemodynamics and NO levels in the ONH were determined. The effect of pretreatment with a NOS inhibitor on the IOP-induced changes was also investigated.

RESULTS. The implicit time of the VEP was prolonged after L-NAME in a dose-dependent manner, whereas the implicit time in the control group (saline) was less affected. Blood flow in the ONH was not reduced by an elevation of the IOP (50 mm Hg) but was significantly reduced by L-NAME (20 mg/kg). The NO metabolites, which were elevated in the ONH during IOP elevation in the control, were also depressed by L-NAME pretreatment.

CONCLUSIONS. These results indicate that NO may play a role in the autoregulation of circulation in the ONH during elevated IOP. This would mean that NO provides some neuroprotection during an acute phase of ischemia in the ONH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Although the retinal vasculature and the vascular system feeding the optic nerve head (ONH) have no innervation, blood flow in these regions is adjusted to the metabolic needs through an autoregulatory mechanism.1 2 The mechanism has still not been determined, although recently, most agree that endothelial cells play an important role in regulating vascular tone by secreting constricting and relaxing factors.

Nitric oxide (NO) has been identified as an endothelium-derived relaxing factor and is synthesized from L-arginine by nitric oxide synthase (NOS). Although NO has been shown to be essential for maintaining the basal current in the retina3 and ONH4 and also for the increase of blood flow in the ONH in response to flickering light stimuli,5 6 the exact role played by NO in the vascular autoregulation under fluctuating perfusion pressure is still controversial. Determining the mechanism for the blood flow alterations in the ONH during fluctuations of the IOP is a key issue for glaucoma therapy, because dysfunction of autoregulation may be involved in the pathogenesis of the glaucomatous alterations.7 8 9 10 11

The techniques used to measure blood flow by viewing the fundus pose difficulties, because the pupillary diameter and corneal transparency are decreased when the IOP is artificially elevated. We hypothesized that a dysfunction of autoregulation can be detected by monitoring the visual evoked potentials (VEPs). Thus, to determine the relationship between NO and vascular autoregulation, we recorded VEPs before and during an elevation of IOP in rabbit eyes in vivo. To investigate whether NO was involved in the autoregulation, a NOS inhibitor was given before the IOP elevation. The hemodynamics of the ONH was measured by the hydrogen clearance method, and the NO levels in the ONH were determined by measuring the levels of nitrite and nitrate, stable metabolites of NO.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
NG-nitro-L-arginine methyl ester (L-NAME) was purchased from Sigma (St. Louis, MO). L-NAME is a structural analogue of L-arginine and inhibits the production of NO through competition with L-arginine.

Animals
Albino rabbits (2.5–3.3 kg) were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). Rabbits were housed in an air-conditioned room at approximately 23°C and 60% humidity with a 12-hour light–dark cycle. All animals were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Recording VEPs
The method of recording VEPs has been described in detail elsewhere.12 13 Briefly, VEPs were elicited from conscious, restrained animals by a photic stimulator (model SLS 4100), amplified with the bandpass filter set at 1.5 to 100 Hz (model AVM-10), and fed to a signal averager (model DAT-1100; all from Nihon-Kohden, Tokyo, Japan). Only the right eye was stimulated in each rabbit for the VEP recordings. The pupil was fully dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P; Santen Pharmaceutical Co., Osaka, Japan), and the animal was dark-adapted for 15 minutes before the recordings. Because there is almost complete decussation in rabbits, the active electrode was placed over the left primary visual area (6 mm anterior and 6 mm to the left of the lambda point),14 15 16 with the reference electrode placed on the midline 16 mm anterior to the lambda point. Grounded was provided by an electrode on the right ear.

The stimulus intensity was 0.6 J, and 32 responses were averaged at a stimulation rate of 1.0 Hz. The stimulated right eye was held open with a Barraquer wire speculum, and the left eye was carefully patched to prevent stray light stimulation. A diffuser was placed before the right eye to ensure Ganzfeld stimulation. The mean luminance at the corneal surface was 0.42 lux/sec.

The analog data were recorded on a rectilinear pen recorder and were also digitized and stored in a microcomputer system for later analysis (MacLab 2e; AD Instruments, Castle Hill, New South Wales, Australia). The implicit time and amplitude of the first negative peak (N1) were measured. Initially, we confirmed that the VEP implicit times and amplitudes were stable and then recorded and stored the baseline values. The experimental procedures were then performed (i.e., an intravenous injection of L-NAME followed by raising the IOP), and the VEPs were recorded every 15 minutes for 60 minutes.

Elevation of IOP
A 25-gauge needle, connected to a bottle of balanced saline solution (BSS Plus; Alcon Laboratories, Fort Worth, TX) or Ringer’s solution by infusion tubing, was inserted into the anterior chamber of the rabbit’s eye under 4% lidocaine local anesthesia. The IOP was increased to 50 mm Hg by increasing the height of the bottle. Pressure was maintained at 50 mm Hg, by monitoring the IOP with a pressure transducer (P10EZ; Gould Statham Instruments, Hatorey, Puerto Rico) throughout the experiment.

Effect of L-NAME on VEP Changes Induced by Elevated IOP
We evaluated the effect of intravenous L-NAME or saline (control, n = 6) on the alterations of the VEPs induced by the elevated IOPs. L-NAME was dissolved in 0.3 mL saline and was injected at a dose of 10 mg/kg (n = 6), 20 mg/kg (n = 6), or 50 mg/kg (n = 6). We also examined the effect of various doses of intravenous L-NAME (10 mg/kg, n = 5; 20 mg/kg, n = 4; 50 mg/kg, n = 7) and saline alone (n = 7) on the VEPs at normal IOPs.

Analysis of Blood Flow in the ONH
Rabbits (n = 12) were anesthetized by intraperitoneal urethane (0.8 g/kg) followed by a continuous intravenous injection of pentobarbital sodium (Nembutal; Abbott Laboratories, Chicago, IL) at a rate of 10 mg/kg·h using a syringe pump (Terufusion TE-311; Terumo, Tokyo, Japan).

The capillary blood flow in the ONH was measured by a hydrogen gas clearance flowmeter (MHG-D1; Unique Medical, Tokyo, Japan). A hydrogen electrode (ON96-045A, platinum needle with a 0.1-mm diameter Pt-Ir tip) was inserted into the lower portion of the ONH from the pars plana, while viewing the fundus through a vitrectomy lens. The reference electrode was fixed in the subcutaneous tissue of the head. After the rabbit inhaled 8% hydrogen gas at a rate of 5.0 L/min for 4 minutes, the capillary blood flow in the ONH was measured by determining the half-life of the hydrogen gas density. This technique has been described in detail elsewhere.17 18

In six rabbits, one eye was selected (three right and three left eyes), and the IOP was artificially elevated to 50 mm Hg. The changes of capillary blood flow in the ONH in response to the IOP elevation was evaluated during the 60 minutes after IOP elevation in the experimental and contralateral normotensive eyes (n = 6). We also examined the effects of intravenous L-NAME (20 mg/kg) on the changes of hemodynamics in the ONH caused by the IOP elevation in another six rabbits.

Measurement of NO Metabolites in the ONH
NO is a short-lived substance and is oxidized to the stable metabolites nitrite and nitrate. To determine the NO levels, it is generally accepted that these metabolites can be assayed.19 Changes of the NO levels in the ONH caused by an IOP elevation were measured by determining the concentration of these metabolites by microdialysis and high-performance liquid chromatography (HPLC) based on the Griess method. This method has been used to detect NO levels in the brain in vivo,20 21 and the procedures are described in detail elsewhere.22 Briefly, a concentric microdialysis probe (15 mm length; A-1-30-O15; Eicom, Kyoto, Japan) was inserted through a guide cannula into the right ONH, where no vessels were visible. Ringer’s solution (140 mM NaCl, 4 mM KCl, l.26 mM CaCl2, and l.15 mM MgCl2; pH 7.4) was perfused at a constant flow rate of 2 µL/min.

The perfused dialysates were collected every 10 minutes in the sample loop of an automated sample injector connected to an automated NO detector-HPLC system (EN0-20; Eicom). Nitrite and nitrate in the dialysate were separated by a reversed-phase separation column packed with polystyrene polymer (NO-PAK, 4.6 x 50 mm; Eicom). The nitrate in the sample was reduced in a cadmium column (NO-RED; Eicom) to nitrite, which reacts with the Griess reagent naphthylethylenediamine to form a purple azo dye, while the nitrite in the sample bypasses the cadmium column for measurements. The levels of nitrite and nitrate in the 10-minute dialysate sample were determined by measuring the absorbance of the color product at 540 nm by a flow-through spectrophotometer (NOD-10; Eicom).

After the basal levels of nitrite and nitrate were measured, the IOP was artificially elevated to 50 mm Hg after an intravenous injection of L-NAME (50 mg/kg, n = 6) or saline (control, n = 7). Ringer’s solution was used to elevate IOP in this part of the experiments, because the oxyglutathione contained in the BSS can affect NO metabolism. Because an earlier ischemia–reperfusion study suggested that NO production was also enhanced in the postischemic stage, we measured the NO levels during the 30 minutes of increased IOP (50 mm Hg) and during the subsequent 30 minutes after normalizing the IOP (15 mm Hg).

Measurement of Blood Pressure
To investigate the effects of L-NAME on general hemodynamics, the blood pressure (BP) was measured in the front leg by an automatic sphygmomanometer (BP-98E; Softron, Tokyo, Japan), which allows a noninvasive measurement of systemic arterial pressure.23 A close correspondence between the pressure determined by the sphygmomanometer and that obtained through a pressure transducer canula placed in the femoral artery has been confirmed previously.24

Statistical Analysis
Unless otherwise noted, data are expressed as the mean ± SD. Two-way interactions were analyzed by repeated-measures analysis of variance (ANOVA) and statistical comparisons between two groups by Student’s t-test. When the data were compared with the baseline levels, a paired t-test was adopted. The level of significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The implicit time and amplitude of the first negative peak (N1) of the VEPs were used to monitor the physiological state, because they showed good reproducibility for individual animals although some variations between rabbits were noted. The mean coefficients of variation (SD/mean) for the implicit time and the amplitude of N1, calculated for each rabbit for five different responses, were 0.46% and 4.19%, respectively. Reduction of the stimulus intensity prolonged the implicit time and reduced the amplitude (data not shown) of N1 of the VEP.

Effect of L-NAME on IOP-Induced VEP Alterations
The VEP implicit times and amplitudes were almost unchanged when the IOP was raised to 50 mm Hg for 60 minutes in the control eyes (Fig. 1A ). In contrast, elevation of the IOP to 50 mm Hg after 50 mg/kg intravenous L-NAME led to a prolongation of the N1 implicit time (Fig. 1B) . Although the implicit time tended to recover slowly, it did not return to baseline until 60 minutes after IOP elevation.



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  		Figure 1. Changes of the visual evoked potentials in an eye pretreated with intravenous 50 mg/kg of L-NAME (B) or saline vehicle control (A) and subjected to an IOP elevation. Downward deflection was negative.

 
Effect of Dosage of L-NAME on IOP-Induced Alterations of VEPs
The changes in the implicit time and the amplitude of the VEPs caused by various doses of intravenous L-NAME are summarized in Table 1 . Because the blood flow in the ONH and retina is affected by the systemic blood pressure, the changes of blood pressure caused by intravenous L-NAME were monitored during these experiments and are summarized in Table 2 .


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  		Table 1. Effects of L-NAME on VEPs in Eyes with Elevated IOP and in Normotensive Eyes

 

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  		Table 2. Blood Pressure Changes Caused by L-NAME in Eyes with IOP Elevation

 
The changes of the VEP implicit times after IOP elevation after treatment with various doses of L-NAME are shown in Figure 2 . The effects of L-NAME on the changes of VEP appeared to be dose dependent. In the control group (IOP elevation without L-NAME), there was a transient prolongation of the implicit time at 15 minutes after IOP elevation; the increase in the implicit time was significant (P = 0.017; paired t-test). The amplitudes were slightly reduced in the control, but the changes were not significant compared with the baseline level (paired t-test).



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  		Figure 2. Changes of visual evoked potential implicit times in rabbit eyes with IOP elevation after pretreatment with intravenous L-NAME at the doses shown. Compared with the vehicle control, the implicit times were significantly increased in groups receiving L-NAME at 20 and 50 mg/kg (P = 0.049 and 0.011, respectively; repeated-measures ANOVA). No significant increase was noted in the 10-mg/kg L-NAME group (P = 0.122; repeated-measures ANOVA). *Significant difference from the control (P < 0.05, Student’s t-test).

 
The same pattern in the changes of implicit time was also seen in the L-NAME–treated groups. However, compared with the control, the VEP implicit times were more prolonged at the higher doses of L-NAME, and its recovery was delayed. Thus, a significant increase in implicit time was seen in the groups given L-NAME at 20 and 50 mg/kg compared with the control (P = 0.049 and 0.011, respectively, ANOVA), whereas no significant difference was found between the group given L-NAME at 10 mg/kg and the control (P = 0.122; ANOVA; Fig. 2 ). Although the N1 amplitude tended to decrease with increasing doses of L-NAME, the differences were not significant (P > 0.05, ANOVA). These changes of VEP induced by L-NAME were not secondary to a reduction of the perfusion pressure, because L-NAME tended to elevate the BP (Table 2) .

The alterations in implicit time caused by L-NAME in eyes with IOP elevation were not due to L-NAME, because L-NAME given to normotensive eyes (IOP = 18.1 ± 3.3 mm Hg) had no effect on the implicit times of VEPs, with a maximum change of 101.8% of baseline at 30 minutes after the L-NAME injection (20 mg/kg; Table 1 ). However, L-NAME also had a tendency to reduce the VEP amplitudes in normotensive eyes (Table 1) .

Effect of L-NAME on IOP-Induced Alterations of Hemodynamics
To evaluate the relationship between the VEP findings and changes in the hemodynamics on the ONH in response to IOP elevation, we measured the capillary blood flow in the ONH under elevated IOP, with and without NOS inhibition. An elevation of IOP to 50 mm Hg alone did not reduce the blood flow significantly in the ONH compared with that in the contralateral eye (P = 0.095, ANOVA; Fig. 3 ). Rather, the blood flow was transiently enhanced at 15 minutes after IOP elevation (P = 0.046, paired t-test; Fig. 3A ).



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  		Figure 3. Alteration of blood flow in the ONH caused by elevation of IOP (A) and its modification by an NOS inhibitor (B). An elevation of IOP to 50 mm Hg alone did not reduce blood flow to the ONH, compared with the contralateral normotensive eye (P = 0.095; repeated-measures ANOVA, A). Although L-NAME (20 mg/kg) alone reduced the basal flow in the ONH and reduced blood flow, even in the normotensive contralateral eyes, inhibition of NOS in the eyes with elevated IOP caused a further significant reduction of blood flow (P = 0.015; repeated-measures ANOVA, B).

 
Although L-NAME (20 mg/kg) reduced the basal blood flow in the ONH, inhibition of NOS in the eyes with elevated IOP caused a further significant reduction of blood flow (Fig. 3B ; P = 0.015; ANOVA). The net changes of blood flow corrected by the values obtained from the contralateral normotensive eye to eliminate systemic factors and the influence of L-NAME on the basal blood flow are shown in Figure 4 . Significant changes were detected between the animals treated and not treated with L-NAME (P = 0.007, ANOVA). These findings demonstrated that blood flow in the ONH was significantly modified by NOS inhibition in the eyes with elevated IOP (Fig. 4) .



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  		Figure 4. Net change of blood flow corrected by the data obtained from the contralateral normotensive eye to eliminate systemic factors and the influence of L-NAME on the basal current. Significant differences were detected between the animals with and without L-NAME (P = 0.007; repeated-measures ANOVA). *Significant differences between those with and without L-NAME (P < 0.05, Student’s t-test).

 
Effect of L-NAME on NO levels in ONH
The baseline levels of nitrite and nitrate in the ONH were 0.72 ± 0.31 and 37.8 ± 12.1 µM, respectively, and the comparable levels in Ringer’s solution were 0.10 ± 0.06 and 4.94 ± 1.19 µM. The differences in the amount of these substances suggests that nitrite in the solution can easily be converted to nitrate.

Changes of nitrate in the ONH relative to the baseline induced by IOP elevation are shown in Figure 5 . The nitrate levels were significantly elevated by increasing the IOP, and a second enhancement was also detected after normalizing IOP to 15 mm Hg. These changes were completely inhibited by L-NAME (P = 0.011, ANOVA; Fig. 5 ). Although the baseline value of nitrite was very low compared with that of nitrate, the changes of nitrite were also significant (P = 0.0496, ANOVA), with maximum 147% at 10 minutes after IOP elevation.



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  		Figure 5. Changes of nitrate levels in the ONH induced by IOP elevation, with and without NOS inhibition. Two-way interaction was significant (P = 0.011; repeated-measures ANOVA). *Significant differences between the animals, with and without L-NAME (P < 0.05, Student’s t-test). Nitrate levels relative to the baseline were increased after IOP elevation and were completely suppressed by L-NAME (50 mg/kg). A second enhancement of nitrate was also detected after IOP was normalized to 15 mm Hg.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that the IOP-induced alterations of the VEP responses and ONH hemodynamics were clearly modified by L-NAME, a NOS inhibitor. Although a transient prolongation of the VEP implicit time was observed in the controls (IOP elevation without L-NAME), the level returned to the baseline value by 45 minutes, suggesting that the autoregulatory mechanism was initially impaired but improved with prolonged elevation of the IOP. Supporting this idea, blood flow in the ONH was not reduced; rather, it was enhanced at 15 minutes after IOP elevation, presumably through autoregulation, to overcome the decreased perfusion pressure. Because inhibition of NOS led to more significant prolongation of the VEP implicit times and reduced ONH blood flow, we conclude that the autoregulatory mechanism was significantly impaired and that NO seems to be essential for the maintenance of the blood supply and visual function in eyes with reduced perfusion pressure. Additionally, an increase of NO metabolites in the ONH was detected 10 minutes after the IOP elevation that paralleled the transient enhancement of blood flow in the control eyes. Taken together, the results strongly suggest that NO is the mediator of the autoregulation—that is, a decrease in the perfusion pressure activates NOS to synthesize NO, which then acts to maintain blood flow and preserve the implicit times of the VEP responses.

A transient prolongation of VEP implicit time was observed in the control eyes at 15 minutes when the blood flow in the ONH was enhanced. This change is difficult to explain, but we suggest that blood flow may be decreased transiently soon after IOP elevation and then may improve. It would require some time for the functional recovery, which may account for the disparity.

Consistent with previous reports indicating that NO may regulate basal blood flow in the choroid25 and the retina,3 26 the basal flow in the ONH was reduced by intravenous L-NAME (Fig. 2B) . L-NAME also had a tendency to decrease VEP amplitudes, even in normotensive eyes. Because the VEP implicit time was not altered significantly by L-NAME in normotensive eyes, we suggest that the reduction of the amplitude of the VEPs caused by L-NAME may indicate impaired retinal function including ganglion cells rather than dysfunction of optic nerve, although the basal flow in the ONH was reduced by L-NAME.

We did not explore the alterations of the electroretinograms (ERGs) and retinal circulation, because the retinal vascular system is poorly developed in rabbits, with blood vessels extending only along the medullary rays. We have shown that a reduction of blood supply to the retinal vasculature by intravitreal endothelin-1 did not affect the b-wave amplitude or the implicit times of the oscillatory potentials of the ERGs—signs of retinal ischemia.27 These results suggest that a large part of the retina is fed by choroidal circulation. Ganglion cells could be the most seriously affected by insufficient choroidal blood flow in rabbits caused by L-NAME. Because autoregulatory mechanisms are generally considered to operate in the ONH and retinal vascular system and not in the choroid, we focused our attention on the alterations of the VEP and ONH blood flow.

The results demonstrated that NOS inhibition during reduced perfusion pressure due to IOP elevation caused deterioration in the function of the eye in the acute phase of retinal ischemia. Constitutive NOS isoforms are expressed by neurons (neuronal NOS) and vascular endothelial cells (endothelial NOS), whereas inducible NOS, activated inflammatory cytokines, is expressed in the retinal pigment epithelium, Müller cells, and vascular smooth muscle cells. The impact of NO on neuronal function and its degeneration under ischemic conditions is very complex. L-Arginine, a precursor of NO, has been shown to improve visual function under ischemic conditions. Hangai et al.28 reported that L-NAME, a selective inhibitor of constitutive NOS, aggravated retinal damage. In contrast, many reports have shown that NO has a neurotoxic effect after ischemia–reperfusion injury, because both L-NAME and NG-(1-iminoethyl)-L-ornithine (a selective inhibitor of inducible NOS) can inhibit the neurotoxic effects of glutamate.29 30 31 Retinal toxicity caused by intravitreal application of an NO donor has been shown in rabbits.32 We are not certain which type of NOS contributed to the enhanced NO production during the IOP elevation in the control eyes. However, because vascular tone is mainly regulated by endothelial NOS, endothelial NOS may have been activated and may have had some neuroprotective effect when the perfusion pressure was decreased, such as occurs during acute angle-closure glaucoma or during vitreous surgery with an increase of the infusion pressure. Consistent with this idea, Neufeld et al.33 also reported an apparent increase in NOS in the ONH of patients with primary open-angle glaucoma. However, in the central nervous system, deficiency of neuronal NOS is known to prevent delayed cell death of neurons after ischemia, whereas endothelial NOS deficiency aggravates ischemic neuronal damage.34 Furthermore, constitutive NOS activation with an increase of intracellular Ca2+ is known to be followed by a later enhancement of inducible NOS activity.35 Once inducible NOS is activated, a large amount of NO is generated, independent of the Ca2+ ion level. NO reacts with superoxide anion and generates peroxynitrite,36 whereas it degrades highly reactive hydroxyl radicals leading to retinal damage. Recently, poly(ADP-ribose) polymerase activation, which is required to repair DNA damage caused by peroxynitrite, has been shown to account for cellular injury, because it leads to rapid depletion of intracellular adenosine triphosphate (ATP) pools.37 Therefore, further long-term studies are needed to analyze the varied and complex effects of NOS inhibitors on ocular ischemia.


   Acknowledgements
 
The authors thank Hideaki Hara and Takashi Ota for technical assistance.


   Footnotes
 
Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan

Submitted for publication July 6, 2001; revised November 5, 2001; accepted November 27, 201.

Commercial relationships policy: N.

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.

Corresponding author: Hidehiro Oku, Department of Ophthalmology, Osaka Medical College, 2-7 Daigaku-cho Takatsuki Osaka, 569-8686 Japan; hidehirooku{at}aol.com


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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