Download Regional regulation of choroidal blood flow by autonomic

Am J Physiol Regulatory Integrative Comp Physiol
279: R202–R209, 2000.
Regional regulation of choroidal blood flow
by autonomic innervation in the rat
JENA J. STEINLE, DORA KRIZSAN-AGBAS, AND PETER G. SMITH
Department of Molecular and Integrative Physiology and R. L. Smith Mental Retardation
Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7401
Received 15 September 1999; accepted in final form 3 February 2000
and interactions apparently determine the level of choroidal perfusion.
Although autonomic innervation may be important
in regulating choroidal blood flow, it is not known
whether sympathetic and parasympathetic nerves determine total choroidal perfusion through interactions
on common vascular beds or whether different types of
innervation predominate in different regions. Questions also remain regarding the extent to which parasympathetic nerves mediate the facial nerve vasodilatory response and the identity of vasodilatory
neurotransmitters. In the present study, we used laser
Doppler flowmetry, which offers a relatively high degree of spatial resolution, to determine whether responses to autonomic nerve stimulation vary within
different regions of the choroid. We have combined this
methodology with discrete stimulation of parasympathetic and sympathetic innervation and selective pharmacological blockade to assess the regional regulation
of choroidal blood flow by autonomic innervation in the
rat.
parasympathetic nervous system; sympathetic nervous system; laser Doppler flowmetry; nitric oxide
be closely regulated to maintain normal retinal function (8). The choroidal blood
vessels, which lie between the outer retina and sclera,
provide much of the retinal perfusion in humans and
other mammals. The choroid contains dense sympathetic innervation originating in the ipsilateral superior cervical ganglion (24). Stimulation of the cervical
sympathetic trunk (CST), which provides preganglionic innervation to the superior cervical ganglion, diminishes choroidal blood flow predominantly by ␣1adrenoreceptor activation (21). The choroid also
receives parasympathetic innervation from the ipsilateral pterygopalatine ganglion (30), which facial nerve
stimulation studies suggest is vasodilatory (35). Therefore, the choroid contains sympathetic vasoconstrictor
and parasympathetic vasodilator nerves whose activity
Studies were conducted on 25 adult female Sprague-Dawley rats (Harlan) aged ⬃60–80 days and weighing 180–240 g.
Rats were anesthetized with urethan (1.25 g/kg ip), rectal
temperature was maintained at 36°C, and a femoral artery
and vein were cannulated for blood pressure recording
(Statham; Costa Mesa, CA) and drug administration, respectively. The distal right facial nerve was exposed and cut to
eliminate movement of the facial musculature. The CST was
exposed through a ventral midline incision in the neck, and a
cuff electrode (Roboz Microprobe; Rockville, MD) was placed
around the intact nerve just caudal to the superior cervical
ganglion for stimulation. The wires were externalized, the
incision was closed with silk suture, and the rat was placed in
a stereotaxic frame (Stoelting; Wood Dale, IL). Surgical procedures and all subsequent experimental manipulations
were approved by the Institutional Animal Care and Use
Committee of the University of Kansas Medical Center.
Parasympathetic and sympathetic stimulation. A scalp incision was made over the sagittal suture, and a craniotomy
was performed. A semi-microbipolar concentric electrode
(100 ␮m contact diameter, Rhodes Medical Instruments;
Woodland Hills, CA) was positioned stereotaxically within
Address for reprint requests and other correspondence: P. G.
Smith, Dept. of Molecular and Integrative Physiology, Univ. of
Kansas Medical Center, Kansas City, KS 66160-7401 (E-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
OCULAR BLOOD FLOW MUST
R202
MATERIALS AND METHODS
0363-6119/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 18, 2017
Steinle, Jena J., Dora Krizsan-Agbas, and Peter G.
Smith. Regional regulation of choroidal blood flow by autonomic innervation in the rat. Am J Physiol Regulatory Integrative Comp Physiol 279: R202–R209, 2000.—Regional influences of parasympathetic and sympathetic innervation on
choroidal blood flow were investigated in anesthetized rats.
Parasympathetic pterygopalatine neurons were activated by
electrically stimulating the superior salivatory nucleus,
whereas sympathetic neurons were activated by cervical
sympathetic trunk stimulation and uveal blood flow was
measured by laser Doppler flowmetry. Parasympathetic
stimulation increased flux in the anterior choroid and nasal
vortex veins but not in the posterior choroid. Vasodilation
was blocked completely by the neuronal nitric oxide synthase
inhibitor 1-(2-trifluoromethylphenyl)imidazole but was unaffected by atropine. Sympathetic stimulation decreased flux in
all regions, and this was blocked by prazosin. Parasympathetic stimulation did not affect vasoconstrictor responses to
sympathetic stimulation in the posterior choroid but attenuated the decrease in blood flow through the anterior choroid
and vortex veins via a nitrergic mechanism. We conclude that
sympathetic ␣-noradrenergic vasoconstriction occurs throughout the choroid, whereas parasympathetic nitrergic vasodilation plays a selective role in modulating blood flow in anterior tissues of the eye.
AUTONOMIC REGULATION OF CHOROIDAL BLOOD FLOW
hydrogen peroxide solution (Sigma; St. Louis, MO). The reaction was terminated by removing the tissue to distilled
water. Endogenous erythrocyte peroxidase activity resulted
in a brown precipitate, which defined the pattern of arterial
and venous vessels. Both methods confirmed that recording
sites showing high-evoked flux levels corresponded to the
vortex veins, an aggregate of vessels with diameters of about
150–250 ␮m. In subsequent physiological experiments, vortex venous recording sites could be consistently identified
based on their nasal location and markedly increased flux
during SSN stimulation.
Recordings from the posterior choroid were obtained by
positioning the probe posterior to the vortex veins. Aside
from the vortex veins, all other recording sites in the posterior choroid appeared to be highly uniform with regard to
basal flux, and none showed any appreciable increase with
SSN stimulation.
Drugs. The following pharmacological agents were dissolved in distilled water and administered through the femoral venous cannula: 1-(2-trifluoromethylphenyl)imidazole
(TRIM, 40–60 mg/kg iv, Research Biochemicals International; Natick, MA), a selective inhibitor of neuronal nitric oxide
synthase (NOS) (14), with the water vehicle slightly acidified
to promote solubility; atropine methyl nitrate (0.5 mg/kg iv,
Sigma Chemical; St. Louis, MO), a muscarinic receptor antagonist; prazosin (1 mg/kg iv, Sigma Chemical), an ␣1adrenoreceptor antagonist. Choroidal blood flow changes
after drug administration were obtained 5–40 min postinjection.
Statistical analysis. Responses to stimulation and drugs
were compared statistically by one-way or repeated-measures
analysis of variance with post hoc comparisons by the Student-Newman-Keuls method and by Student’s t-test (SigmaStat 2.03, Jandel Scientific; Corte Madera, CA). All values
are presented as means ⫾ SE, and P ⬍ 0.05 was considered
statistically significant.
RESULTS
Vortex veins. Basal blood flux recorded from the
nasal vortex veins showed 0.2- to 0.3-Hz oscillations of
low amplitude (Fig. 1), which were not synchronous
with respiration and may reflect spontaneous uveal
vasomotor activity reported by others (29). During SSN
stimulation, peak flux increased one- to twofold (Figs. 1
and 2, P ⫽ 0.015), reaching a maximum within 20 s
that was maintained until stimulation was stopped
after 40–60 s. Flux values returned to baseline levels
in ⬃20–40 s. Although the oscillation frequency did not
change during stimulation, amplitude increased substantially (Fig. 1). SSN stimulation did not alter systemic blood pressure (Table 1).
Atropine did not influence basal flux or systemic
blood pressure (Table 1), and the increase in flux during SSN stimulation was not affected (Fig. 2). The
selective neuronal NOS inhibitor TRIM caused a transient decrease in systemic arterial blood pressure and
choroidal flux, both returning to baseline within 3 min
(Table 1). TRIM blocked the response to SSN stimulation (P ⫽ 0.004 vs. stimulation before TRIM; not significant vs. prestimulation, Fig. 2).
In a separate group of rats, CST stimulation caused
vortex venous flux to decrease significantly without
obvious effects on amplitude or frequency of oscillations. CST stimulation decreased vortex venous flux by
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 18, 2017
the superior salivatory nucleus [SSN, 9.5 mm posterior, 9.5
mm ventral, 2.5 mm lateral to bregma (27)], as described
previously (2, 22, 32). This nucleus provides the preganglionic innervation to the parasympathetic pterygopalatine
ganglion (34). Parasympathetic innervation was activated by
electrical stimulation of the SSN at 12–20 Hz, 3 V for 40–60 s
(Grass SD9 stimulator; Quincy, MA), which we have shown
previously to elicit maximal activation of orbital parasympathetic innervation (2); this was confirmed for blood flow in
preliminary experiments. Electrode placement within the
preganglionic parasympathetic nucleus was confirmed by
porphyrin discharge from the harderian gland during stimulation (36) and by electrolytic lesioning at the end of the
experiment followed by examination of cresyl violet-stained
frozen sections to verify electrode position within the SSN as
described previously (2).
Ocular sympathetic innervation was stimulated using the
cuff electrode placed around the preganglionic CST. Stimulations were performed at 12–20 Hz, 30 V for 40–60 s, which
is supramaximal for sympathetic activation (2, 33), as confirmed for blood flow changes in preliminary investigations in
the present study.
Blood flow measurements. Blood flow was measured using
laser Doppler flowmetry (floLAB model with MP3 probe,
Moor Instruments; Devon, UK). This technique is based on
the concept that laser light reflected from a moving object,
such as a red blood cell, undergoes a Doppler frequency shift
that is determined by the relative concentration of blood cells
and their average velocity (28). Flux, which is directly proportional to blood flow (28), was recorded (time constant of
0.5 s) on a computer using Polyview software (Astro-Med,
Grass Instruments). Previous studies using this technique
have shown linear flow measurements for vessels with diameters similar to those from which measurements were obtained in the present study (17).
To assess blood flow in the anterior choroid, a flow probe (1
mm tip diameter) was positioned extraocularly ⬃3 mm distal
to the limbus, as described by others (18). To obtain recordings from the posterior choroid and the vortex veins, the
pupil was dilated by topical application of a 0.01% solution of
epinephrine, the cornea was incised, the fiberoptic probe was
inserted using a micromanipulator through the anterior and
posterior chambers and the vitreous body, and mineral oil
was applied to prevent corneal drying. Because of technical
constraints, it was not possible to sample all intraocular
regions. Therefore, recordings were restricted to the nasal
postequatorial (hereafter referred to as nasal) hemisphere.
At the end of the experiment, background flux was recorded
following circulatory arrest induced by anesthetic overdose.
In preliminary experiments the relationship between flux
and vascular topography was explored. Flux characteristics
were recorded from different intraocular regions. Some posterior nasal regions were consistently found to have high
parasympathetically evoked flux levels and were readily distinguished from all adjacent regions that showed no consistent response to SSN stimulation. These sites were assessed
anatomically by two methods. Some rats were killed by
anesthetic overdose with the probe in situ, and the sclera was
excised to expose the recording site. In others, the recording
site was determined either by using a suture tie as a landmark or by advancing the probe to induce local trauma and
erythrocyte extravasation. The vasculature was then visualized by a peroxidase reaction. Eyes were removed following
circulatory arrest and immersion fixed in 4% buffered formaldehyde for 2 h. After fixation, the anterior chamber was
removed, and the remainder of the eye was placed for 1 h in
a 24-well plate with 1 mg/1 ml diaminobenzidine in 0.005%
R203
R204
AUTONOMIC REGULATION OF CHOROIDAL BLOOD FLOW
Fig. 1. Laser Doppler flux recordings from the anterior
choroid, posterior choroid, and the nasal vortex veins
during stimulation of either the superior salivatory nucleus (SSN) or the cervical sympathetic trunk (CST). All
recordings were obtained from a single rat with the
exception of the anterior choroid. Responses obtained
from the vortex veins and anterior choroid during CST
stimulation were similar to that shown for the posterior
choroid. Bar indicates period of SSN stimulation (20 Hz,
3 V) or CST stimulation (12 Hz, 30 V).
was stimulated alone (Fig. 4). Irrespective of the order
of stimulation, activation of the second site attenuated
the effect of the first. SSN stimulation reduced the
CST-induced decrease in flux to ⫺11.5 ⫾ 2.5% of prestimulation baseline, and CST stimulation attenuated
the SSN-induced increase in flux to ⫺7.7 ⫾ 5% of
baseline. Flux during sympathetic and parasympathetic costimulation was not significantly different
from prestimulation basal flux [Fig. 4, not significant
by paired t-test].
Costimulation after TRIM administration produced
a decrease in flux that was significantly greater than
that obtained in the absence of TRIM (P ⬍ 0.001) and
was not significantly different from that during CST
stimulation alone (Fig. 4). Administration of atropine
following TRIM did not elicit any further change (Fig.
4). Prazosin, when given after TRIM and atropine,
prevented any significant change in flux (Fig. 4, P ⬍
0.001 vs. CST stimulation prior to prazosin, not significantly different from prestimulation flux).
Posterior choroid. In another group of rats prepared
for costimulation with the flow probe positioned to
record from the posterior choroidal vasculature, actiTable 1. Mean arterial blood pressures
SSN stimulation
CST stimulation
Costimulation
Atropine
TRIM
Prazosin
Fig. 2. Effect of parasympathetic SSN stimulation on vortex venous
flux under control conditions and after the administration of atropine (0.5 mg/kg) and 1-(2-trifluoromethylphenyl)imidazole (TRIM,
40–60 mg/kg). Data are displayed as means ⫾ SE for a group of 5
rats. A, P ⬍ 0.05 vs. prestimulation flux; B, not significant vs. control;
C, P ⬍ 0.01 vs. control and atropine; D, not significant vs. prestimulation flux.
Before
During
91 ⫾ 6
90 ⫾ 10
82 ⫾ 5
91 ⫾ 6
86 ⫾ 6
75 ⫾ 7
93 ⫾ 6
88 ⫾ 10
84 ⫾ 6
82 ⫾ 8
83 ⫾ 7
53 ⫾ 1*
Values are means ⫾ SE for 8 rats in all categories except superior
saluatory nucleus (SSN) stimulation (n ⫽ 10) and prazosin (n ⫽ 6).
Mean arterial blood pressure (mmHg) is measured before and during
neuronal stimulation and in the presence of drugs in different groups
of rats. SSN stimulation was conducted at 12–20 Hz, 3 V. Cervical
sympathetic trunk (CST) stimulation was conducted at 12–20 Hz, 30
V. Drug dosages were 0.5 mg/kg atropine, 40–60 mg/kg 1-(2-trifluoromethylphenyl)imidazole (TRIM), and 1 mg/kg prazosin, and all
measurements were obtained ⬃5–10 min after intravenous administration. * P ⱕ 0.001 vs. before prazosin.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 18, 2017
⬃60% (Fig. 3, P ⫽ 0.002). CST stimulation did not
affect systemic arterial blood pressure (Table 1). The
␣-adrenoceptor antagonist prazosin significantly attenuated the CST stimulation-induced decrease (Fig.
3, P ⬍ 0.001 vs. stimulation before prazosin), and flux
during stimulation was not significantly different from
prestimulation basal flux. However, prazosin induced a
marked decrease in systemic arterial pressure (Table
1) with an accompanying decrease in basal flux.
To assess interactions between parasympathetic and
sympathetic innervation on vortex venous flux, another group of rats was prepared for SSN and CST
costimulation. In these rats, either the SSN or CST
was stimulated for 20 s, after which stimulation of the
other site commenced in combination with the initial
stimulus. Stimulations of both sites were maintained
for a total 60 s, with measurements obtained 40 s after
costimulation commenced. Maximal changes in vortex
venous flux were essentially identical to those obtained
in previous experiments in which one site or the other
AUTONOMIC REGULATION OF CHOROIDAL BLOOD FLOW
R205
and the posterior choroid (Fig. 6). However, flux values
during costimulation were significantly greater in the
anterior choroid than in the posterior choroid (P ⬍
0.001, Fig. 6). Administration of TRIM before costimulation resulted in a decrease in flux that was comparable to that obtained from the vortex veins and was
significantly different from flux changes in response to
costimulation alone (P ⬍ 0.001). Atropine did not affect
the response to costimulation. Prazosin effectively
blocked the remaining change in flux (Fig. 6).
DISCUSSION
vation of the SSN alone resulted in no consistent effect
on posterior choroidal blood flux (Figs. 1 and 5, P ⬍
0.005 vs. vortex vein response). The absence of any
apparent effect of SSN stimulation persisted in all
accessible regions of the posterior choroid with the
exception of those containing the vortex veins. In contrast, CST stimulation decreased posterior choroidal
flux in a fashion similar to that observed for the vortex
veins (Figs. 1 and 5). Costimulation yielded flux
changes that were comparable to those obtained from
the posterior choroid with CST stimulation alone (Fig.
5) and were significantly less than flux changes recorded from the vortex veins during costimulation (P ⬍
0.001). Because parasympathetic stimulation exerted
no effects on flux, neither TRIM nor atropine was
administered. Prazosin abolished all effects of CST
stimulation (Fig. 5).
Anterior choroid. In transscleral recordings of the
anterior choroid, SSN stimulation caused an increase
in flux that was similar to that recorded from the
vortex veins and was significantly greater than posterior choroid flux (Figs. 1 and 6, P ⫽ 0.010). CST stimulation elicited a decrease in flux that was comparable
in magnitude to those observed in both the vortex veins
Autonomic regulation of choroidal perfusion. Stimulation of parasympathetic preganglionic neurons of the
SSN elicits a pronounced increase in flux within the
vortex veins. Because these vessels represent the primary route of choroidal blood efflux, this finding is
consistent with increased overall blood flow throughout the choroid and associated tissues. Furthermore,
because this was not accompanied by changes in systemic blood pressure, it is most consistent with a decrease in vascular resistance due to vasodilation. These
findings are in agreement with previous studies showing increased choroidal blood flow and microsphere
accumulation during facial nerve stimulation (35).
Moreover, because increased flow was elicited by selectively stimulating preganglionic parasympathetic neurons, our findings provide strong evidence that vasodilation accompanying facial nerve stimulation is
mediated by parasympathetic axons and not by other
fiber populations [e.g., sensory, sympathetic (23), or
somatic motor] that also travel in this nerve. We therefore conclude that a parasympathetic nerve pathway
originating in the SSN and with postganglionic fibers
traveling in the facial nerve is a potent modulator of
uveal blood flow.
Although previous microsphere studies provide evidence of parasympathetically mediated choroidal vasodilation, regional influences were not examined. Unlike microsphere analyses, laser Doppler flowmetry
can readily measure flow in relatively discrete volumes
of tissue (⬃1.5 mm3) (1) and therefore is well suited for
assessing regional differences in changes to perfusion.
Fig. 4. Changes in vortex venous flux in response to
stimulation of the parasympathetic SSN alone (SSN
stimulation), stimulation of the CST (CST stimulation),
and simultaneous stimulation of both parasympathetic
and sympathetic innervation (costimulation). Venous flux
responses during costimulation were assessed following
administration of TRIM (40–60 mg/kg, TRIM ⫹ costimulation), atropine (0.5 mg/kg, atropine ⫹ TRIM ⫹ costimulation), and prazosin (1.0 mg/kg, prazosin ⫹ atropine ⫹
TRIM ⫹ costimulation). Data are displayed as means ⫾
SE for 5 rats in all groups except costimulation, which
was 6, and included experiments using both stimulation
sequences. A, P ⬍ 0.005 vs. prestimulation; B, P ⬍ 0.001
vs. SSN or CST stimulation; C, not significant vs. prestimulation;, D, P ⬍ 0.001 vs. costimulation; E, not significant vs. CST stimulation; F, not significant vs.
TRIM ⫹ costimulation; G, P ⬍ 0.001 vs. CST stimulation
and atropine ⫹ TRIM ⫹ costimulation.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 18, 2017
Fig. 3. Effect of CST stimulation on vortex venous flux under control
conditions and after the administration of prazosin (1.0 mg/kg). Data
are displayed as means ⫾ SE for 5 rats. A, P ⫽ 0.002 vs. prestimulation flux; B, P ⬍ 0.001 vs. control; C, not significant vs. prestimulation flux.
R206
AUTONOMIC REGULATION OF CHOROIDAL BLOOD FLOW
Fig. 5. Changes in posterior choroidal flux in response to
SSN stimulation, CST stimulation, and costimulation.
Posterior flux responses during costimulation were obtained after the administration of prazosin (1.0 mg/kg,
prazosin ⫹ costimulation). Data are displayed as
means ⫾ SE, with SSN stimulation obtained from 6 rats,
CST stimulation from 5 rats, and costimulation and
prazosin from 3 rats. A, P ⬍ 0.001 vs. prestimulation; B,
P ⬍ 0.001 vs. SSN stimulation; C, P ⬍ 0.001 vs. costimulation and CST stimulation and not significant vs. baseline flux.
Fig. 6. Changes in anterior choroid flux in response to
SSN stimulation, CST stimulation, and costimulation.
Anterior choroid flux responses were measured after the
administration of TRIM (40–60 mg/kg, TRIM ⫹ costimulation), atropine (0.5 mg/kg, atropine ⫹ TRIM ⫹ costimulation), and prazosin (1.0 mg/kg, prazosin ⫹ atropine ⫹
TRIM ⫹ costimulation). Data are displayed as means ⫾
SE for 4–5 rats in each treatment. A, P ⬍ 0.005 vs.
prestimulation; B, P ⬍ 0.001 vs. SSN or CST stimulation;
C, not significant vs. prestimulation; D, P ⬍ 0.001 vs.
costimulation, E, not significant vs. CST stimulation; F,
not significant vs. TRIM ⫹ costimulation; G, P ⬍ 0.001
vs. CST stimulation and atropine ⫹ TRIM ⫹ costimulation.
ary arteries are strongly influenced by pterygopalatine
parasympathetic innervation, whereas those of the
short ciliary and central retinal artery vessels are
appreciably less affected. The absence of detectable
parasympathetic influences on posterior choroidal flow
is somewhat surprising in light of immunohistochemical studies showing NOS-immunoreactive nerves in
the posterior choroid of the rat (39) and pigeon (4).
Nonetheless, these fibers may simply be axons of passage en route to more anterior targets, or the posterior
choroidal vascular smooth muscle may be relatively
insensitive to released parasympathetic neurotransmitters.
Functionally, these findings may provide insight as
to the role of parasympathetic innervation. The anterior tissue from which we recorded likely contains some
anteriorly projecting blood vessels from posterior choroid but is probably dominated by branches from the
major arterial circle and pars plana venules that drain
the more anterior tissues (supplied by the long posterior and anterior ciliary arteries), particularly the ciliary body and processes (10). These structures are
integrally involved in formation of aqueous humor.
Because aqueous humor formation is determined in
part by the extent of perfusion of the anterior uveal
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 18, 2017
This technique provides clear evidence of major regional differences in parasympathetic modulation of
choroidal flow. Thus, whereas the nasal vortex veins
showed substantial flux increases during parasympathetic stimulation, all other sampled regions of posterior choroid (even as close as 1 mm to the vortex veins)
showed little to no change in perfusion during stimulation. In contrast, the anterior choroid showed large
increases in flux. We conclude that increased vortex
venous flux during parasympathetic stimulation is primarily due to increased flow in anterior vasculature
without detectable changes in posterior choroidal flow.
These findings therefore may imply that different neural control mechanisms exist within different ocular
regions.
These striking regional differences in parasympathetic regulation may reflect differences in origin and
function of the choroidal vasculature. Anterior and
posterior ocular structures derive their primary blood
supplies from different sources. In the rat, the posterior choroid is perfused by the short posterior ciliary
and central retinal arteries, whereas the anterior uvea
is perfused by the long posterior and anterior ciliary
arteries (6, 9). It is therefore probable that the arterioles supplied by the long posterior and anterior cili-
AUTONOMIC REGULATION OF CHOROIDAL BLOOD FLOW
Recordings of flux from the vortex veins show that
under normal conditions, flow during sympathetic and
parasympathetic costimulation is similar to or slightly
below prestimulation levels. Because the posterior choroid is markedly vasoconstricted during costimulation,
vortex venous outflow may reflect a relative predominance of parasympathetic vasodilation in the anterior
choroid, and indeed flux during costimulation is significantly greater in the anterior choroid than in the posterior choroid. Therefore, whereas sympathetic vasoconstriction is not detectably affected by parasympathetic
activation in the posterior choroid, it appears to be negated by parasympathetic nerves in the anterior region.
Parasympathetic nerves can nullify sympathetic actions through two potential mechanisms. Target cell
activity may reflect equal or preferential responsivity
to parasympathetic transmitters or these neuronal
populations can interact prejunctionally, with parasympathetic nerves inhibiting sympathetic transmitter release. This latter mechanism is particularly important in many target systems. Parasympathetic
nerves inhibit sympathetic neurotransmitter release
via prejunctional muscarinic receptors in a variety of
organ systems, including the heart, vasculature, vas
deferens, and periorbital smooth muscle (2, 25, 37, 38).
If this mechanism is important in regulating choroidal
perfusion, then parasympathetic stimulation during
sympathetic activation in the neuronal NOS-inhibited
preparation should still attenuate sympathetic vasoconstriction and this should be countered by the administration of atropine. However, our findings from
anterior choroidal and vortex venous recording sites
show that NOS inhibition completely blocks the parasympathetic effects and that atropine has no significant actions. Therefore, prejunctional muscarinic
inhibition of sympathetic neurotransmission by parasympathetic nerves apparently is not an important
mechanism in regulating choroidal blood flow.
It is unclear why this mechanism, which is widespread
in the peripheral nervous system, is not operative in the
choroid. One possibility is that sympathetic neurons that
project to the choroid lack prejunctional muscarinic receptors. Although a high proportion (65–85%) of superior
cervical ganglion neurons express high-affinity muscarinic binding sites (16), we cannot rule out the possibility
that choroidal sympathetic nerves lack muscarinic receptors. Alternatively, even though both parasympathetic
and sympathetic axons travel together within Schwann
cells in some parts of the choroid (31), their terminals
may be segregated, such that acetylcholine released by
parasympathetic nerves is unable to activate prejunctional muscarinic receptors. In any event, it appears that
autonomic projections to the choroid display a high degree of functional segregation as revealed by their selective regional distribution and absence of prejunctional
muscarinic interactions.
Perspectives
This study provides evidence that regional differences exist in the autonomic regulation of blood flow in
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 18, 2017
structures, increased blood flow via a parasympathetic
projection may provide a selective mechanism for modulating perfusion of these tissues. Indeed, it is well
established that stimulation of the facial nerve increases intraocular pressure (11), as does stimulation
of diencephalic regions (12) that project to the SSN
[e.g., lateral hypothalamus (34)]. These findings therefore are accordant with the view that a parasympathetic pathway from the SSN via the pterygopalatine
ganglion plays an important role in regulating anterior
uveal perfusion.
Neuronal NOS mediates parasympathetic choroidal
vasodilation. Previous studies have shown that facial
nerve vasodilation is atropine resistant and therefore
not likely to be cholinergic (3), a finding confirmed in
the present experiments. In light of the potent vasodilatory effects of NO in other systems (13), this neurotransmitter would be a logical candidate. Indeed, nonselective NOS inhibitors decrease basal choroidal blood
flow in pigs, dogs, and rats (7, 15, 20) and attenuate the
increased flow during facial nerve stimulation in the
rabbit (26). However, because nonselective NOS inhibitors elevate basal systemic blood pressure, thereby
increasing choroidal perfusion, it has been unclear
whether other transmitters (e.g., vasoactive intestinal
polypeptide) may also participate in parasympathetically mediated vasodilation (26). In the present study,
the highly selective neuronal NOS inhibitor TRIM (14)
did not affect systemic blood pressure or basal uveal
flow, a finding similar to that reported for another
neuronal NOS antagonist, 7-nitro indazole (7-NI) (20).
However, TRIM completely abolished effects of parasympathetic stimulation on anterior choroidal and vortex venous blood flow, even at the relatively high stimulation frequencies used in these studies. Although
this is the first study to document the involvement of
NO in neurally evoked uveal vasodilation of the rat,
similar findings were obtained in the pigeon, where
vasodilation mediated by oculomotor nerve efferents
was eliminated by 7-NI (40). However, the selectivity of
this NOS inhibitor has been questioned recently (5). In
contrast to previous studies using nonselective NOS
inhibitors (19), TRIM in the present study did not
affect basal flux at doses that completely blocked vasodilation during parasympathetic nerve stimulation.
These findings support the idea that parasympathetic
nitrergic mechanisms do not play an appreciable role
in determining basal choroidal blood flow but are responsible for choroidal vasodilation that accompanies
parasympathetic stimulation in the anesthetized rat.
Interactions between parasympathetic and sympathetic innervation in the regulation of choroidal perfusion. Together with previous studies, our findings show
that sympathetic innervation exerts potent ␣1-adrenoceptor-mediated vasoconstriction in the anterior (18)
and posterior choroid, whereas parasympathetic vasodilation influences primarily anterior uveal tissues.
Because both sympathetic and parasympathetic
nerves regulate anterior perfusion, blood flow in this
region should reflect the summation of the interactions
between these populations.
R207
R208
AUTONOMIC REGULATION OF CHOROIDAL BLOOD FLOW
This work was supported by the National Institute of Child
Health and Human Development Grant HD-33025, with core support from center Grant HD-02528.
15.
16.
17.
18.
19.
20.
21.
22.
REFERENCES
1. Barnett NJ, Dougherty G, and Pettinger SJ. Comparative
study of two laser Doppler blood flowmeters. J Med Eng Technol
14: 243–249, 1990.
2. Beauregard CL and Smith PG. Parasympathetic innervation
of rat peri-orbital smooth muscle: prejunctional cholinergic inhibition of sympathetic neurotransmission without direct postjunctional actions. J Pharmacol Exp Ther 268: 1284–1288, 1994.
3. Bill A and Sperber GO. Control of retinal and choroidal blood
flow. Eye 4: 319–325, 1990.
4. Cuthbertson S, Jackson B, Toledo C, Fitzgerald ME, Shih
YF, Zagvazdin Y, and Reiner A. Innervation of orbital and
choroidal blood vessels by the pterygopalatine ganglion in pigeons. J Comp Neurol 386: 422–442, 1997.
5. Cuthbertson S, Zagvazdin YS, Kimble TD, Lamoreaux WJ,
Jackson BS, Fitzgerald ME, and Reiner A. Preganglionic
endings from nucleus of Edinger-Westphal in pigeon ciliary ganglion contain neuronal nitric oxide synthase. Vis Neurosci 16:
819–834, 1999.
6. Davson H. Physiology of the Eye. New York: Pergamon, 1990.
7. Deussen A, Sonntag M, and Vogel R. L-Arginine-derived
nitric oxide: a major determinant of uveal blood flow. Exp Eye
Res 57: 129–134, 1993.
8. Foulds WS. The choroidal circulation and retinal metabolism–
an overview. Eye 4: 243–248, 1990.
9. François J and Neetens A. Comparative anatomy of the vascular supply of the eye in vertebrates. In: The Eye: Comparative
Physiology, edited by Davson H and Graham LT Jr. New York:
Academic, 1974, p. 1–70.
10. Funk RH. The vessel architecture of the pars plana in the
cynomolgus monkey, rat and rabbit eye. A scanning electron
microscopic study of plastic corrosion casts. Ophthalmic Res 25:
337–348, 1993.
11. Gloster J. Influence of the facial nerve on intra-ocular pressure.
Br J Ophthalmol 45: 259–278, 1961.
12. Gloster J and Greaves DP. Effect of diencephalic stimulation
upon intra-ocular pressure. Br J Ophthalmol 41: 513–532, 1957.
13. Gustafsson LE, Wiklund CU, Wiklund NP, Persson MG,
and Moncada S. Modulation of autonomic neuroeffector transmission by nitric oxide in guinea pig ileum. Biochem Biophys Res
Commun 173: 106–110, 1990.
14. Handy RL, Harb HL, Wallace P, Gaffen Z, Whitehead KJ,
and Moore PK. Inhibition of nitric oxide synthase by 1-(2-
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
trifluoromethylphenyl) imidazole (TRIM) in vitro: antinociceptive and cardiovascular effects. Br J Pharmacol 119: 423–431,
1996.
Jacot JL, O’Neill JT, Scandling DM, West SD, and McKenzie JE. Nitric oxide modulation of retinal, choroidal, and anterior uveal blood flow in newborn piglets. J Ocul Pharmacol Ther
14: 473–489, 1998.
James S and Burnstock G. Autoradiographic localization of
muscarinic receptors on cultured, peptide-containing neurones
from newborn rat superior cervical ganglion. Brain Res 498:
205–214, 1989.
Kajiya F, Tsujioka K, Ogasawara Y, Hiramatsu O, Wada Y,
Goto M, and Yanaka M. Analysis of the characteristics of the
flow velocity waveforms in left atrial small arteries and veins in
the dog. Circ Res 65: 1172–1181, 1989.
Kawarai M and Koss MC. Sympathetic vasoconstriction in the
rat anterior choroid is mediated by alpha1-adrenoceptors. Eur
J Pharmacol 363: 35–40, 1998.
Kelly PA, Buckley CH, Ritchie IM, and O’Brien C. Possible
role for nitric oxide releasing nerves in the regulation of ocular
blood flow in the rat. Br J Ophthalmol 82: 1199–1202, 1998.
Koss MC. Role of nitric oxide in maintenance of basal anterior
choroidal blood flow in rats. Invest Ophthalmol Vis Sci 39:
559–564, 1998.
Koss MC and Gherezghiher T. Adrenoceptor subtypes involved in neurally evoked sympathetic vasoconstriction in the
anterior choroid of cats. Exp Eye Res 57: 441–447, 1993.
Krizsan-Agbas D, Zhang R, Marzban F, and Smith PG.
Presynaptic adrenergic facilitation of parasympathetic neurotransmission in sympathectomized rat smooth muscle. J Physiol
(Lond) 512: 841–849, 1998.
Kuwayama Y, Grimes PA, Ponte B, and Stone RA. Autonomic neurons supplying the rat eye and the intraorbital distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactivity. Exp Eye Res 44: 907–922, 1987.
Laties AM and Jacobowitz D. A comparative study of the
autonomic innervation of the eye in monkey, cat, and rabbit.
Anat Rec 156: 383–395, 1966.
Muscholl E. Peripheral muscarinic control of norepinephrine
release in the cardiovascular system. Am J Physiol Heart Circ
Physiol 239: H713–H720, 1980.
Nilsson SF. The significance of nitric oxide for parasympathetic
vasodilation in the eye and other orbital tissues in the cat. Exp
Eye Res 70: 61–72, 2000.
Paxinos G and Watson C. The Rat Brain in Stereotaxic Coordinates. San Diego, CA: Academic, 1986.
Riva CE, Cranstoun SD, Mann RM, and Barnes GE. Local
choroidal blood flow in the cat by laser Doppler flowmetry. Invest
Ophthalmol Vis Sci 35: 608–618, 1994.
Riva CE, Pournaras CJ, Poitry-Yamate CL, and Petrig BL.
Rhythmic changes in velocity, volume, and flow of blood in the
optic nerve head tissue. Microvasc Res 40: 36–45, 1990.
Ruskell GL. An ocular parasympathetic nerve pathway of facial
nerve origin and its influence on intraocular pressure. Exp Eye
Res 10: 319–330, 1970.
Ruskell GL. Facial parasympathetic innervation of the choroidal blood vessels in monkeys. Exp Eye Res 12: 166–172, 1971.
Smith PG and Beauregard CL. Conversion of parasympathetic nerve function from prejunctional inhibition to postjunctional excitation following sympathectomy of rat periorbital
smooth muscle. Brain Res 629: 319–322, 1993.
Smith PG, Evoniuk G, Poston CW, and Mills E. Relation
between functional maturation of cervical sympathetic innervation and ontogeny of ␣-noradrenergic smooth muscle contraction
in the rat. Neuroscience 8: 609–616, 1983.
Spencer SE, Sawyer WB, Wada H, Platt KB, and Loewy
AD. CNS projections to the pterygopalatine parasympathetic
preganglionic neurons in the rat: a retrograde transneuronal
viral cell body labeling study. Brain Res 534: 149–169, 1990.
Stjernschantz J and Bill A. Vasomotor effects of facial nerve
stimulation: noncholinergic vasodilation in the eye. Acta Physiol
Scand 109: 45–50, 1980.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 18, 2017
the eye. Specifically, the posterior choroid is dominated
by sympathetic vasoconstrictive input, whereas parasympathetic nitrergic vasodilatory nerves preferentially regulate flow in anterior tissues. Because many
ocular disease states are associated with regional disturbances in ocular perfusion, it is interesting to speculate as to possible roles of autonomic innervation in
the etiology of these diseases. Moreover, manipulation
of either sympathetic or parasympathetic innervation
may selectively impact different tissues, depending on
their autonomic profile. Indeed, it may be predicted
from these studies that vascular parasympathetic innervation plays an important role in regulating perfusion of the anterior tissues, including those associated
with aqueous humor formation. It is particularly interesting therefore that NOS inhibitors are proving effective in clinical trials at combating elevated intraocular
pressure associated with glaucoma. Whether other
therapeutic strategies can also take advantage of regional selectivity of ocular autonomic effects remains
to be determined.
AUTONOMIC REGULATION OF CHOROIDAL BLOOD FLOW
36. Tashiro S, Smith CC, Badger E, and Kezur E. Chromadacryorrhea, a new criterion for biological assay of acetylcholine. Proc
Soc Exp Biol Med 44: 658–661, 1940.
37. Vanhoutte PM and Levy MN. Prejunctional cholinergic
modulation of adrenergic neurotransmission in the cardiovascular system. Am J Physiol Heart Circ Physiol 238: H275–
H281, 1980.
38. Westfall DP, Millecchia LL, Lee TJF, Corey SP, Smith DJ,
and Fleming WW. Effects of denervation and reserpine on
R209
nexuses in the rat vas deferens. Eur J Pharmacol 41: 239–242,
1977.
39. Yamamoto R, Bredt DS, Snyder SH, and Stone RA. The
localization of nitric oxide synthase in the rat eye and related
cranial ganglia. Neuroscience 54: 189–200, 1993.
40. Zagvazdin YS, Fitzgerald ME, Sancesario G, and Reiner A.
Neural nitric oxide mediates Edinger-Westphal nucleus evoked
increase in choroidal blood flow in the pigeon. Invest Ophthalmol
Vis Sci 37: 666–672, 1996.
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 18, 2017