Retinal Vessel Diameter Responses to Central Electrical Stimulation in the Rat: Effect of Nitric Oxide Synthase Inhibition

Abstract

Purpose: Recent histological data suggest autonomic innervation of the central retinal artery. In the present study, we investigated the effect of electrical brain stem stimulation at the superior salivatory nucleus (SSN) on the retinal vessel diameter in rats and whether nitric oxide mediates a possible effect.

Methods: Sprague-Dawley rats (n = 12) were anesthetized using pentobarbital sodium (50 mg/kg intraperitoneally). The animals were artificially ventilated and the femoral artery and vein were cannulated for blood pressure measurement and drug administration. After a craniotomy was performed, a unipolar stainless steel electrode was inserted into the brainstem at the coordinates of the SSN. Stimulations were performed at 20 Hz, 9 μA, 1 ms pulse duration and 200 pulses. Retinal vessel diameters were measured continuously with the Imedos DVA-R, a noncontact fundus camera for rats with image analysis software. After control measurements, L-NAME, a nonspecific inhibitor of NO synthase, was applied intravenously (10 mg/kg), and the SSN stimulations were repeated.

Results: Stimulation at the SSN coordinates increased the retinal arterial diameter by 6.41% ± 1.65% and the venous diameter by 3.48% ± 1.93% (both P < 0.05). Application of L-NAME reduced the arterial response significantly to 2.93% ± 0.91%, but did not change the venous response. Mean arterial pressure, carotid blood flow, and heart rate remained unaltered (by the stimulation).

Conclusions: The present study demonstrates that the retinal circulation reacts to electric stimulation at the SSN coordinates in rats. Nitric oxide is involved in the response, but it is not the sole neurotransmitter.

An adequate blood supply to the retina is crucial for its function and thereby vision. To ensure this adequate supply over a wide range of ocular perfusion pressures, retinal blood flow is actively regulated.¹ The current understanding is that the retinal circulation is under mainly myogenic and metabolic control.² As opposed to the uveal vasculature, the intraocular retinal vessels lack innervation from the autonomic nervous system^3,4 and, consequently, retinal vascular tone was assumed to be controlled by local mechanisms influenced by the local metabolism. More recent histological investigations, however, revealed the presence of peptitergic (vasoactive intestinal polypeptide, calcitonin gene–related peptide), nitrergic as well as parasympathetic and sympathetic nerves in the prelaminar section of the central retinal artery (CRA) in rats and monkeys.^5,6 A similar pattern is likely present in humans as well.⁷ A study reporting an increase in retinal blood flow after electrical stimulation of the parasympathetic part of the facial nerve in rabbits was published by Nilsson,⁸ but in this species, the retinal arteries are innervated.^9,10 Other studies by Toda et al.^11,12 indicated the presence of a nitrergic vasodilator tone in retinal vessels in dogs that was abolished by ethanol injection in the pterygopalatine ganglion and isolated monkey CRA preparations dilated in response to transmural electrical stimulation. These studies led to the assumption that the retinal circulation, if not directly innervated, does react to autonomic input. 

In the present study, we tested the hypothesis that the retinal circulation reacts to electrical stimulation of a parasympathetic brain stem nucleus supplying the eye (superior salivatory nucleus [SSN]^13,14) in rats. 

Methods

The study was approved by the Governmental Animal Experiments Committee in Salzburg. All experiments were conducted in accordance with the ARVO guidelines for animal use in vision research. All animals were euthanized with an anesthetic overdose at the end of the experiment without regaining consciousness. 

Animal Preparation

Male Sprague-Dawley rats (n = 12, 293.4 ± 8.6 g) (Charles River Laboratories, Sulzfeld, Germany) were anesthetized using pentobarbital sodium (Sigma-Aldrich, Vienna, Austria; 50 mg/kg intraperitoneally [IP], supplemented intravenously [IV] as needed) and paralyzed using gallamine triethiodide (1 mg/kg, IV). A tracheotomy was performed and the animal was respired with room air (SAR-830 small animal ventilator; CWE, Inc., Ardmore, PA, USA). The preset weight-adjusted respirator settings were adapted to keep the end-tidal CO2 between 35 and 45 mm Hg (CapStar 100 CO2 Analyzer; CWE, Inc.). A catheter for blood pressure measurement (PE50 with a PE10 tip; Becton Dickinson, Sparks, MD, USA) was inserted into the right femoral artery and connected to a pressure transducer (MLT 0380; AD Instruments, Colorado Springs, CO, USA). Another catheter (PE10) was inserted into the right femoral vein for drug administration. The right eye was used for all measurements. The pupil was dilated with mydriatic eye drops (Tropicamid 0.5%; Landesapotheke Salzburg, Austria) and sodium hyaluronate eye drops (Hylocomod; Croma Pharma, Leobendorf, Austria) were used to keep the tear film stable and the cornea moist. 

Superior Salivatory Nucleus Electrode Preparation

After the initial preparation, the animal was fixed in a stereotaxic head holder (Model 900; David Kopf Instruments, Tujunga, CA, USA). Through a skin incision at the cranial midline, the skull was exposed and the Bregma point was calculated as described by Paxinos and Watson.¹⁵ The SSN coordinates were calculated with regard to the Bregma position, 10.6 mm posterior, 2.2 mm lateral, and 9.5 mm below the bone surface (for a schematic drawing of the relevant anatomy and connections of the SSN refer to Refs. 13, 14). With a motorized tungsten-carbide drill (3.2-mm tip diameter), the bone was removed until the dura mater was visible. A layer of bonewax was applied to prevent bleeding once the dura mater was punctured. Then the stimulation electrode was advanced to the calculated coordinates. The height of the electrode was adjusted slightly in some animals until discharge from the harderian gland was clearly visible during the stimulation.¹⁶ To confirm the position of the electrode, the stimulation site was marked in some animals by creating an electric lesion (800 μA, 30 seconds), followed by standard histology staining (cresyl violet; 16-μm thickness). The lesion site was identified by disintegrated and loosened cell structures when compared with the contralateral unlesioned side (Fig. 1). 

Figure 1

 

Schematic drawing of the brain sections. (A) Most caudal section where the facial nerve was fully depicted, Bregma: 10.2 mm. (B) Bregma: 10.68 mm. (C) Sagittal section providing an overview of the anatomy and the approximate positions of sections (A) and (B). The asterisk in (B) marks the site of the lesion as identified by loosened cell structures and cell debris. Reference structures are named following the nomenclature of Paxinos and Watson.¹⁵ 4v, fourth ventricle; 7n, facial nerve; g7, genu of the facial nerve; sp5, spinal trigeminal tract; ml, medial lemniscus; mcp/icp, middle/inferior cerebellar peduncle.

Retinal Vessel Diameter Measurement

The retinal vessel diameters were measured continuously with a digital noncontact fundus camera adapted to provide fundus images of rat eyes (DVA-rodent; Imedos UG, Jena, Germany). The technical setup has been published by Link et al.¹⁷; therefore, only a short overview is given here. A noncontact flood illumination fundus camera for rats is used in combination with a 50-W Xenon lamp. For retinal vessel imaging with maximized contrast, the illumination spectrum was filtered at 560 ± 15 nm. The imaging was performed with a 0.5-inch charge-coupled device camera (752 × 582 pixels) (CF 8/5 MX; Kappa opto-electronics GmbH, Gleichen, Germany) and recorded digitally using a frame grabber (25 frames/second) (Solius eX; Matrox Imaging, Dorval, Quebec, Canada). The diameter of a retinal artery and vein were measured continuously with the Imedos RVA Research software (RVA Research 4.16; Imedos UG).¹⁸ The software allows vessel sections up to 500 μm in length within a region of interest set by the operator to be measured. For the measurement, one artery and one vein starting at the rim of the optic nerve head were chosen. Note that although the operator can choose the region of interest and designate the vessels to be measured, the software selects the actual measurement segment (within the region of interest) where the vessel boundaries can be detected reliably. 

The instrument measures the vessel diameter in measurement units, which can be converted to an absolute size measurement based on the reproduction scale of the instrument. However, this conversion is based on an idealized model of a rat eye (analogous to Gullstrand's eye model), whereas the actual conversion factor depends on the optical properties of the individual eye, as well as on the optical properties of the fluid meniscus applied through eye drops. Measurement units can be converted to a micrometer reading to estimate the order of magnitude of the diameters but not for an exact determination. 

For these reasons (variability in exact measurement location, magnification factor), all measurements of retinal vessel diameters have been normalized and the change in relation to the baseline value is given. 

Experimental Protocols

Because the experimental setup involves a tear film meniscus on the cornea prone to evaporation and therefore a change in the optical properties of the system, we performed tests on the stability of the setup. Continuous measurements over a period of 10 minutes were performed and the drift and coefficient of variation of the mean arterial pressure (MAP), heart rate (HR), and arterial and venous diameter measurements assessed (n = 12). 

The time course of the reaction of the retinal circulation as well as the systemic parameters was determined in the second protocol. A nonspecific inhibitor of nitric oxide synthase (NOS), L-NG-Nitroarginine methyl ester (L-NAME; 10 mg/kg IV), was applied after a baseline measurement period of 1 minute and MAP, heart rate (HR), and vessel diameters were measured continuously (n = 5). 

Superior Salivatory Nucleus Stimulation Protocol (n = 7)

After a stable baseline period for all measured variables, the SSN was stimulated electrically using a unipolar electrode (5710; A&M Systems, Carlsborg, WA, USA) and a current controlled stimulator (FE180 Isolated Stimulator; AD Instruments). Stimulations were performed with a pulse train of 200 current pulses at an amplitude of 9 μA, and a pulse duration of 1 ms with frequency of 20 Hz. This brief, 5-second stimulation period was chosen to minimize depletion of neurotransmitter reserves and possible damage to tissue at the electrode tip. 

The same protocol was used after application of L-NAME (10 mg/kg, bolus IV). In some animals, the position of the stimulation electrode tip had to be adjusted vertically in the range of 100 to 200 μm to regain the stimulation effect. After the application of L-NAME (and subsequent SSN stimulations), hexamethonium chloride (20 mg/kg, IV), a ganglionic blocker, was applied and the electrical stimulation was repeated. 

Data Analysis

All parameters were recorded continuously on a digital recording system (PowerLab; AD Instruments), connected to a standard personal computer. The recording software package (LabChart 7, v7.2.5; AD Instruments) was used to determine the mean values of the measured variables during the minutes of stable baseline before stimulation and then during and after the response to the stimulation. 

Results are given as mean ± SEM. Baseline and stimulation values for all parameters were compared using repeated measures ANOVA with post hoc paired t-tests and Student-Newman-Keuls correction, with P < 0.05 considered significant (Systat Software, Chicago, IL, USA). 

Results

The Table shows the results of the baseline variability measurements. Over a 10-minute period, all measured variables drifted less than 1%. The coefficient of variation is in the order of magnitude of 1% for all parameters. 

Table.

 

Evaluation of the Optical Stability of the Setup

 

Figure 2 shows the time change in vessel diameters after the application of L-NAME (10 mg/kg, IV), as well as the change in arterial blood pressure. The application of L-NAME without SSN stimulation resulted in a decrease in the arterial diameter of 6.52% ± 1.59% and the venous diameter declined by 1.86% ± 0.75%; the MAP rose by 45.14% ± 12.24%. 

Figure 2

 

Time course of the response of MAP and retinal arterial and venous diameters after the application of an IV bolus of 10 mg/kg L-NAME. After 5 minutes, there is no significant change in blood pressure or arterial diameter, while the venous diameter continuous to decline. The asterisk indicates a significant change against baseline (minute 1).

Figure 3 shows the reaction of the retinal vessels to electrical stimulation of the SSN at control conditions, after L-NAME application and after hexamethonium chloride application. Stimulation at the SSN coordinates increased the retinal arterial diameter by 6.41% ± 1.65% and the venous diameter by 3.48% ± 1.93% (both P < 0.05). Mean arterial pressure and heart rate remained unaltered by the stimulation (99.03 ± 5.66 mm Hg to 98.54 ± 4.77 mm Hg, 279 ± 17 beats per minute to 282 ± 15 beats per minute). Application of L-NAME increased the baseline MAP from 99.03 ± 12.68 mm Hg to 148.97 ± 8.48 (P < 0.001) and reduced the arterial response significantly to 2.93% ± 0.91% (P < 0.05), but did not change the venous response significantly (3.48% ± 1.93% to 3.94% ± 1.52%, P = 0.55). Hexamethonium chloride reduced the stimulation effect to 0.75% ± 0.24% (P = 0.36) for the arteries and −0.43% ± 0.44% (P = 0.12) for the veins. The MAP was reduced by hexamethonium application at baseline, but remained unchanged during SSN stimulation (135.65 ± 15.9 mm Hg and 135.40 ± 14.2 mm Hg, respectively). 

Figure 3

 

Vessel diameter changes in response to SSN stimulation at control conditions, after L-NAME application (10 mg/kg, IV) and after hexamethonium chloride application (20 mg/kg, IV). L-NAME significantly reduces the vasodilation in the retinal artery, while the venous effect remains unchanged. Hexamethonium abolished the stimulation effect. The asterisk denotes P ≤ 0.05 compared with SSN stimulation at control conditions.

Discussion

The retina provides the unique opportunity to directly monitor blood vessels of the microcirculation and retinal vessel diameter analysis is a well-established tool for the assessment of the microcirculation in humans.^19,20 In rats, a special fundus camera allows the continuous measurements of vessel diameters with a noncontact setup.¹⁷ Before testing the study hypothesis, we evaluated the drift of the setup to measure vessel diameters over a longer time span and found the preparation to be stable with a drift less than 1% over 10 minutes (Table). 

Retinal blood flow is known to be under myogenic and metabolic control.^1,21,22 Because retinal vessels themselves are not innervated, it is assumed that retinal blood flow is not influenced by the (autonomic) nervous system.^3,4 To the best of the authors' knowledge, the current literature provides only sparse data on a possible effect of the autonomic nervous system on the retinal circulation, with conflicting results, possibly due to differences in species and experimental methods used. Work by Toda et al.¹¹ with dogs suggests the presence of a neuronal tone in the retinal vessels that is abolished by ethanol injection into the pterygopalatine ganglion. Nilsson et al.⁸ reported an increase in retinal blood flow to stimulation of the parasympathetic nerves to the eye in an article investigating the choroidal blood flow response to parasympathetic stimulation in rabbits. However, rabbits have innervated retinal blood vessels and might not be an appropriate (i.e., representative for the human situation) model to study neuronal influence in the retinal circulation.^9,10 Menage et al.²³ investigated the sympathetic innervation in cynomolgus monkeys and reported no change in retinal blood flow after superior cervical ganglionectomy. 

The main finding of the present study was that the retinal arterial diameters increase significantly in response to electrical brain stem stimulation at the coordinates of the SSN in rats (Fig. 3). Our data do not provide information concerning the origin of the nervous influence (i.e., whether the diameter increase occurs as a result of the innervation of the prelaminar part of the rat CRA or as a secondary effect of a change in blood flow in the ophthalmic artery). Shear stress-induced vasodilation as a reaction to increased blood flow in the preocular CRA or ophthalmic artery might provide an explanation for the results, but this is speculation. The presence of neuronal NOS (nNOS)-positive nerve fibers (among others) in the rat CRA provides an anatomical basis for a direct effect on the CRA (preocular part, with a secondary reaction of the intraocular vessels).⁵ The change in venous diameter might be a reaction to an increase in blood flow, if passive behavior is assumed. It is important to note that the current setup measures the retinal vascular diameter, but not retinal blood flow or the flow in a retinal branch vessel. Hence, the present data indicate only that the retinal circulation does react to SSN stimulation and the vascular resistance does likely decline, but it does not give an estimation of retinal blood flow. Further studies are needed to answer that question and measurements based on Doppler optical coherence tomography are most promising, as first reports are being published.²⁴ 

Nitric oxide is a vasodilator and mediates the effects of various vasoactive substances.²⁵ The effects of NOS inhibition on the retinal circulation are well studied,^26,27 but data in rats have not been reported to date. Therefore, we performed a protocol investigating the systemic and local effects of NOS inhibition in rats, using intravenous application of L-NAME. 

The application of L-NAME without SSN stimulation resulted in a decrease in the arterial diameter of 6.52% ± 1.59% and the venous diameter declined by 1.86% ± 0.75%; the MAP rose by 45.14% ± 12.24%. One study done in humans using a different NOS inhibitor (L-NMMA, 3 mg/kg–6 mg/kg) reported a dose-dependent constriction of the arteries by 2.1% to 3.8% and 5.3% to 7.9% for the veins, alongside a 6% to 11% increase in blood pressure.²⁸ The more pronounced increase in blood pressure in the rat might partly explain the differences found in the arterial vasoconstriction, but our data do not provide a conclusive explanation for the difference in venous effects. 

In the SSN stimulation protocol, L-NAME application reduced the SSN stimulation effect in the artery from 6.42% ± 1.65% to 2.93% ± 0.91%, but did not fully block the dilation response. This is in keeping with data examining the parasympathetic influence on choroidal blood flow, as NOS inhibition reduced but did not fully block the stimulation response in cats and rabbits.^8,29,30 Given the costaining with vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), and other neuropeptides in rats, this finding seems plausible.⁵ It is important to note that L-NAME is a nonselective NOS inhibitor (even though some differences in the inhibition constant exist) and it is not possible to discriminate between endothelial NOS (eNOS)- and nNOS-induced effects. Because L-NAME also increases arterial blood pressure and constricts retinal vessels at baseline, a change of the working point of the vessels (in terms of myogenic autoregulation) can be assumed, and it remains unclear if the decrease in the stimulation effect is a direct effect of NOS inhibition or a secondary effect of its baseline effects. Moreover, retinal vessels are considered within the blood-retina barrier and it is unclear whether IV application is the appropriate route of application. At least for the central nervous system, there is evidence that L-NAME crosses the blood-brain barrier effectively.³¹ The retrolaminar part of the CRA appears to have a less tight blood-retina (brain) barrier as well.³² Hence, we assumed that IV application is appropriate for studying the retinal circulation, as we wanted to avoid the secondary effects of intravitreal application (i.e., changes in IOP and thereby perfusion pressure). 

Hexamethonium chloride is a nicotinic cholinergic receptor antagonist and thereby blocks the signal transmission in autonomic ganglia. In the SSN stimulation protocol, hexamethonium abolished the stimulation effect (Fig. 3), thereby verifying that the effect is mediated by the autonomic nervous system. 

In summary, the present study demonstrates that the retinal circulation does react to autonomic stimulation at the SSN in rats. Inhibition of NOS inhibition attenuates but does not abolish the stimulation effect. Further studies are needed to determine the effect on retinal blood flow and the site of influence. 

Acknowledgments

Supported by the Fuchs-Foundation, the PMU Research Fund (S-12/01/005-STR and E-11/14/071-REI), and the Adele Rabensteiner Foundation. 

Disclosure: C.A. Strohmaier, None; K. Motloch, None; C. Runge, None; A. Trost, None; B. Bogner, None; A. Kaser-Eichberger, None; F. Schrödl, None; M. Lenzhofer, None; H.A. Reitsamer, None