(Stroke. 1998;29:212-221.)
© 1998 American Heart Association, Inc.
Weakness of Sympathetic Neural Control of Human Pial Compared With Superficial Temporal Arteries Reflects Low Innervation Density and Poor Sympathetic Responsiveness
Rosemary D. Bevan, MD;
John Dodge, MS;
Patricia Nichols, DVM;
Paul L. Penar, MD;
Carrie L. Walters, MD;
Terry Wellman, BS;
John A. Bevan, MD
From the Totman Laboratory for Human Cerebrovascular Research (R.D.B.,
J.D., P.N., N.T.-T., T.W., J.A.B.); the Department of Pharmacology, Division
of Neurosurgery (P.L.P.), University of Vermont, Given Building, Burlington,
Vt; and Neurological Surgeons, Phoenix, Az (C.L.W.).
Correspondence to John A. Bevan, MD, Department of Pharmacology, University of Vermont, 303B Given Bldg, Burlington, VT 05405-0068.
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Abstract
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Background and Purpose—The primary goal of these studies was
to understand and investigate the capacity of perivascular nerves to
influence the tone of human pial arteries and to compare them with
other human cephalic arteries, the superficial temporal and middle
meningeal.
Methods—Responses to electrical activation of intramural nerves
and related features of fresh segments of human cephalic arteries—the
pial (PA; 478±34 µm ID), middle meningeal (MMA; 540±41
µm ID), and superficial temporal (STA; 639±49 µm
ID)—obtained from patients aged 15 to 82 years during surgical
procedures were studied on a resistance artery myograph.
Results—The PA segment responses to electrical nerve activation
and to norepinephrine (NE; 10-5 mol/L) were
1% and 21% of tissue maximum, respectively, compared with 6% and
34% for the MMA and 14% and 90% for the STA. Tissue maximum was
defined as the force increase to 127 mmol/L KCl plus arginine
vasopressin (1 µm). All arteries dilated well to acetylcholine.
Possible explanations for the PA marginal neurogenic responses were
assessed. NE ED50 was 5.4±2.2x10-7 mol/L and
did not vary with age or diameter. NE responsiveness did not increase
in vessels with spontaneous or raised potassium-induced tone.
Relaxation to isoproterenol was variable and
propranolol did not increase the neurogenic response.
Neither
NG-monomethyl-L-arginine,
NG-nitro-L-arginine methyl
ester, endothelium removal, nor
indomethacin consistently influenced the
contractions to NE or neurogenic reactivity. The weak PA neurogenic
response is in keeping with its poor innervation. As determined by
catecholamine histofluorescence, innervation in the
PA is sparse, with density increasing in the order PA, MMA, and STA.
The incidence of nerve structures in the PA adventitio-medial junction
was only 3% of those in the STA, and these were situated more than
3 µm from the closest smooth muscle cell.
Conclusions—We conclude that the weak neurogenic response of
adult human pial artery reflects its poor innervation and
responsiveness to NE, implying that these features are not important in
the regulation of its diameter.
Key Words: adrenergic responses • pial artery • sympathetic innervation • temporal arteries
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Introduction
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Much of the
mammalian vasculature receives a sympathetic innervation
that contributes to the control of its caliber at rest and to the
changes that occur in response to stress.1 However, the
role of this innervation in regulation of the cerebral blood flow in
humans is unclear. Although the presence of adrenergic perivascular
nerves has been described in the cerebral arteries of a number of
laboratory animals including humans,2 for some time there
has been much debate about the level of functional control of this
innervation (see, for a few pertinent references, references 3 to 6).
This is particularly the case in humans since in vivo studies are
limited to animals. In vitro preparations of larger arteries from many
species are responsive to electrical perivascular nerve activation, but
remarkable species variation in the magnitude and the direction of the
response has been reported.2 7 Both the presence8 9 10 and absence11 of neurogenic
responses of the larger human cerebral arteries have been observed. In
the only study to date of human pial arteries, no response attributable
to the innervation was observed.12 This is, perhaps, not
surprising since neurogenic control in the rabbit decreases with
decreasing diameter.13 However, in this latter study of
human pial arteries no attempt was made to exclude functional factors
that might mask or minimize a potential response or to understand its
basis. In addition, since neurogenic contraction varies, at least in
the rabbit, with diameter13 and since in humans there are
age-related changes in cerebral vascular function,14 15 16
these were not excluded by these authors as explanations for the
paucity of the responses sought.
In view of the remarkable species variation, an assessment of the
extent of neurogenic control of these arteries in humans cannot be
established by extrapolation from other species. The majority of prior
studies on the human have been carried out on autopsy material at
various times after death. In this paper on human PA we report on our
assessment of the level of neurogenic control of fresh vessels (180 to
700 µm ID) obtained from patients 15 to 82 years old, average
41.5 years, obtained during neurosurgical procedures. Artery segments
were studied in a resistance artery myograph, and nerves were excited
by EFS. Several approaches were used to establish appropriate voltage
parameters for the pial segments, and their effectiveness
was demonstrated in a parallel study of human STA and MMA, some
segments of which came from the same patients. To make this more
inclusive of the human cephalic circulation, MMA segments were also
included. In the initial experimental series the PA segments, in
contrast to those of the STA, were essentially unresponsive to nerve
activation. Responses from MMA were intermediate. For this reason a
number of possible processes that might compromise or mask a
contractile neurogenic response were assessed: weak responsiveness to
NE mediated by 
-adrenoceptors, concurrent ß-adrenoceptor
occupation, absence of basal tone, concurrent stimulation of dilator
nerves, release of nitric oxide
(NO)–endothelium-derived relaxing factor (EDRF),
dilator prostanoids, and other endothelial factors. Our
results showed that these did not provide an explanation for the
absence of responsiveness. The size of the NE responsiveness did not
change significantly with age over the range of 15 to 82 years or with
diameter. Fluorescence histochemistry of the sympathetic
catecholamine perivascular innervation showed a plexus of
low density in the PA and quantitative electron microscopy, a low
density of perivascular nerve bundles widely separated from the outer
layers of the smooth muscle cells. These features were in contrast to
those seen in the STA and seemed to provide an explanation for the
absence of the nerve effect.
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Methods
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Human PA segments were obtained during neurosurgical procedures
from the pial circulation of normal regions of the cerebral cortex, MMA
from the dura mater, and from the STA and its branches. This study was
approved by the Ethics Committee of the University of Vermont. The
patients selected were clinically free of
cardiovascular and metabolic disease.
Arterial segments were obtained (from patients aged: 15 to
82 years for PA, n=23; 18 to 46 years for MMA, n=15; 19 to 75 years for
STA, n=15) and were either studied on the same day of removal or after
storage for 24 hours at 4°C in Krebs'
physiological solution (PSS) (composition in
mmol/L: NaCl 130, KCl 4.7, KH2PO4 1.18,
MgSO4 1.17, NaHCO3 14.9, EDTA 0.026, and
dextrose 11.0) and CaCl2 1.6 containing
deferoxamine (100 µmol/L), heparin (10 U/mL),
penicillin (50 U/mL), and streptomycin (50 µg/mL). Similar functional
results were obtained with the two groups of human tissues.
Preservation for 24 hours under these conditions has been shown
previously not to affect nerve, muscle, or endothelial
function of animal arteries.17 The human tissues were
transferred to the laboratory and prepared for in vitro study. Only
segments free of adherent blood were studied.
Functional Studies
PA, MMA, and STA were cut into two to four rings 3- to 4-mm long
and suspended on wires in resistance artery myographs.18 19
They were suspended in Krebs' PSS maintained at pH=7.4±0.1, 37°C,
and gassed continuously with 95% O2/5% CO2.
The rings were connected to force transducers (model FT03, Grass
Instruments) to record changes in isometric force. Internal
diameter was measured using a video camera (Colorado video
micrometer). The myograph mounting wires were slowly
separated until a barely significant change in the force record was
observed. Wire separation was taken to be half the unstretched
circumference. Platinum wire electrodes (0.3 mm diameter, 3
mm long) placed on either side of the suspended segments were used for
transmural EFS using a Grass stimulator. The internal diameters of PA,
MMA, and STA used in the initial series of studies were 478±34,
540±41, and 639±49 µm, respectively. The STA segment diameters
were significantly larger than those of the MMA or PA
(P<.05). Diameters measured in this way correspond to
unstretched diameter.
Each vascular segment was equilibrated for 90 minutes in PSS maintained
at 37°C and gassed continuously with 95% O2/5%
CO2. The bathing solution was changed every 15 to 20
minutes. Because comparable anatomic artery segments from different
patients varied in their internal diameter, wall thickness, and
elasticity, the active length-tension relationship was determined for
each segment before the experimental protocol. The rings were first
stretched to an internal diameter known from experience to be less than
the optimum and then were exposed to 30 mmol/L KCl. A step
increase in passive wall tension of approximately 10% was then
achieved by stretch and when equilibrium was reached, rings were
exposed to KCl. The preload when the contractile response to KCl was
within 20% of the prior response was considered to provide optimum
length. The rings were then allowed to equilibrate for a further 30
minutes. The arterial segments were exposed to NE (10
µmol/L), and at the contractile plateau ACh (10 µmol/L) was
added to assess the function of the endothelium. A
vessel was included in the experimental series if the relaxation
induced by ACh was >50% of the pre-addition NE-induced tone. When
similar experiments were conducted on more than one segment from the
same patient, results were averaged.
Nerve Stimulation
Neurogenic responses of PA, MMA, and STA segments to transmural
EFS of their intramural nerves were investigated. Various frequencies
were applied as biphasic, square pulses of 0.3-milliseconds' duration
at supramaximal voltage through platinum wire electrodes placed on
either side of the arterial segment and connected to a
Grass stimulator. A pulse duration was adopted of 0.3 millisecond,
which in our experience with blood vessels from various species
provides optimum selectivity of stimulation of neurons but not smooth
muscle cells.1 Two approaches were used to determine
stimulation voltage for selective activation of perivascular nerves,
breakthrough and subtraction (see Fig 2
). Only one method was used in a
particular ring.
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Figure 2. Responses of human PA mounted in a resistance
artery myograph to EFS of intramural nerves using two techniques,
breakthrough and subtraction: for details see "Methods." In
breakthrough, a voltage just short of that causing direct smooth muscle
stimulation is used. In subtraction, responses to the voltages that
concurrently activate both nerve and muscle are reduced by the
muscle response. TTX was used in both instances to reversibly
inactivate the nerves.
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Breakthrough Method
A voltage was used that was less than that required to cause an
increase in wall force in the presence of TTX (3x10-7
mol/L). TTX is considered to block neuronal conduction effectively and
selectively. Thirty minutes after the addition of TTX, EFS was applied
at 10-minute intervals at increasing voltages until a force response
was recorded. This voltage reduced by one volt was used after TTX
washout for the EFS.
Subtraction Method
At 10-minute intervals EFS was applied for 2 minutes at 5, 7.5,
10, 12.5, 15, and 20 V. In some experiments, pulses of 0.6 and 0.3
millisececonds' duration were used. TTX (3x10-7 mol/L)
was added to the tissue bath, and the stimulation train
parameters that caused contraction before TTX addition were
repeated. Differences in the force responses at the same stimulation
parameters are considered to represent responses to
activation of the perivascular innervation. In both methods, trains of
biphasic square wave pulses of 0.3 milliseconds' duration were
delivered to the electrodes at 8 and 16 Hz until the response reached
equilibrium (0.5 to 2 minutes).
To test for dilator neurogenic responses, after the voltage for EFS had
been determined by either the breakthrough or the subtraction method in
the absence of tone, EFS was applied in the presence of tone, either
spontaneous or induced by PGF2 (3x10-6
mol/L). EFS was often repeated in the presence of phentolamine
(10-6 mol/L).
Frequency-response curves were determined from trains of stimuli
delivered at 1, 2, 4, and 8 Hz in random sequence and finally at 16 Hz
until an equilibrium response was derived. Responses to 8 Hz were
elicited at the beginning and end of the series to assess time- and
stimulation-dependent changes. These were found to be minimal.
Cumulative NE concentration-response curves of PA, MMA, and STA were
obtained by adding increasing concentrations of NE (0.01 to 30
µmol/L) to the bath in half-log–unit steps. Each addition was made
only after the response to the previous concentration had reached
equilibrium. The effective concentration (EC50, mol/L) of
NE that causes 50% of maximum contraction (Emax) was
determined. Relaxation to ACh (10 µm) was expressed as %
pre-addition tone.
A variety of other procedures or conditions were used. The extent of
maximum relaxation that occurred on exposure to papaverine
(10–5 to 3x10–5 mol/L) was considered basal
tone. In experiments designed to test the influence of raised tone on
NE responses to PA this was achieved by increasing the K+
concentration in the PSS. To assess possible ß-adrenoceptor
influences, after tone had been increased with PGF2
(10–6 mol/L), PA were exposed to isoproterenol
(3x10–6 to 10–5 mol/L).
Propranolol (10–6 mol/L) was used to show
possible ß-adrenoceptor involvement during EFS. Tissues were exposed
to L-NNA (10–4 mol/L) or L-NAME (3x10–4
mol/L) for 30 minutes to inactivate NO synthesis. The
endothelium was removed or inactivated by
gentle rubbing with a hair. The effectiveness of the procedure was
assessed by loss of dilation to ACh (10–6 mol/l). Exposure
to indomethacin (10–5 mol/L) for 30
minutes was used to inhibit the possible synthesis of dilator
prostanoids.
At the end of each experiment, the arterial segments were
maximally contracted with 127 mmol/L KCl-Krebs' solution plus
arginine vasopressin (1 µmol/L). This is defined as
Emax.
Structural Studies
Catecholamine Nerve Histofluorescence
Segments adjacent to those used for the functional studies were
processed for viewing of catecholaminergic perivascular
nerves using the glyoxylic acid method20 plus pontamine sky
blue (0.5% wt/vol) to mask the nonspecific background
fluorescence21 and were viewed with a Zeiss
epifluorescence microscope.
Electron Microscopy
Segments of arteries 1 to 2 mm in length were
immersion-fixed in 2.5% glutaraldehyde and 2%
paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4,
for 4 hours at room temperature or overnight at 4°C. After a
phosphate buffer rinse, they were postfixed in 2% osmium tetroxide in
phosphate buffer for 1 hour at room temperature. The segments were then
dehydrated in graded alcohols and embedded in Durcupan ACM (Fluka).
Ultrathin transverse sections were cut on a RMC Ultramicrotome (MT-7)
with a diamond knife and stained with uranyl acetate and lead citrate.
The sections were viewed in a JEOL 100 CXII electron microscope at 60
kV.
For the quantitation of nerves, the entire tunica adventitia of each
artery was examined for the presence of nerve bundles. Nerve bundles
are defined as contiguous groups of nerve axons ensheathed in Schwann
cell processes. A bundle may be seen to contain nerve axons
(nonvaricose) and varicosities with synaptic vesicles and mitochondria.
Only entire bundles rather than individual nerve axons or varicosities
were counted in this study. Low-magnification micrographs were taken to
measure the perimeter of the adventitio-medial border. Higher
magnification micrographs were made of each nerve bundle and were used
to measure the distances separating the nerve bundle from the nearest
smooth muscle cell; montages were taken for nerve bundles at greater
distances, and to determine nerve densities, the number of nerve
bundles per unit length of adventitio-medial border. Measurements of
all micrographs were made using the Sigma-Scan measurement system with
a Numonics Digitizing Tablet and cursor linked to a computer.
Drugs Used
The following drugs were used: ACh hydrochloride, NE bitartrate,
indomethacin (Sigma), ± isoproterenol HCl (Sigma),
L-NAME (Sigma), L-NNA (Sigma), DL-propranolol
HCl (Sigma), TTX (Sigma), and arginine vasopressin (Bachem). Drugs were
dissolved in Krebs' solution, prepared freshly every day, and kept on
ice.
Data Analysis
Contractile responses were expressed either as a percentage of
the maximum tension produced by 127 mmol/L KCl-Krebs' and
arginine vasopressin (1 µmol/L) or as active wall tension
divided by the length of the individual arterial segment
and the internal diameter (mg/µm per millimeter). The latter
parameter was used in place of wall thickness because of
the difficulty in making accurately the latter measurement on a tissue
that would subsequently be used for in vitro study. Some of the
segments available to us were short. It was assumed that wall
thickness–to-lumen ratio remains constant over the small range of
vessel diameter used in this study. The unstretched wall
thickness–to-lumen ratio in the PA was approximately 6 (unpublished
data, R.D. Bevan and J.A. Bevan). Values were expressed in relation to
internal diameter to minimize the effect of changes in medial muscle
mass associated with changes in diameter. Concentration-response curves
based on a Hill relationship were fitted to individual
concentration-response data by a computer program (least-square
method).
Agonist potencies were calculated on the basis of data from individual
vessels and are expressed as pD2=-logEC50,
where EC50 is the concentration of the agonist needed to
produce 50% of the maximal response.
Statistical Analysis
Data are given as mean±SEM. In all experiments, n refers to the
number of patients from whom the blood vessels were obtained.
Statistical differences between the mean values were determined by
ANOVA followed by a Scheffé's F test. A value of
P=.05 was accepted as significant for differences between
groups. The relationship between parameters was evaluated
by linear regression analysis of the data using a commercially
available statistical analysis software (Statview)
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Results
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The primary goal of these studies was to understand and
investigate the capacity of perivascular nerves to influence the tone
of human pial arteries and to compare them with other human cephalic
arteries, the superficial temporal and middle meningeal. Because the
initial series of experiments showed only a marginal response of PA to
nerve stimulation and a poor response to NE, subsequent experiments
were carried out in an attempt to account for this. For this reason,
results are presented in two sections—first, a comparison of
the PA, MMA, and STA, and their general characteristics of response to
nerve activation and NE and second, an analysis of the basis of
the poor responsiveness of the PA.
Comparison of the Responsiveness of the Human PA, MMA, and
STA
Maximum Contractile Responsiveness
The maximum change in wall force (Emax) produced by
127 mmol/L KCl-Krebs' solution combined with arginine vasopressin
(1 µmol/L) was 2.76±0.57 mg/µm per millimeter (n=15) in the
STA, 0.90±0.14 mg/µm per millimeter (n=15) in the MMA, and
0.92±0.07 mg/µm per millimeter (n=23) in the PA. (See "Methods"
for basis of expressing wall force.) The STA force development was
significantly greater than in the PA and MMA.
Responses to Exogenous NE
NE (0.01 to 30 µmol/L) produced sustained
concentration-dependent increases in wall tension in the three types of
human arteries tested. Higher concentrations either did not cause
additional force increase or resulted in relaxation. NE was equipotent
in the different groups of arteries, with ED50 values of
5.4±2.2x10–7 mol/L (n=21), 1.1±0.057x10–6
mol/L (n=14), and 1.7±0.29x10–6 mol/L (n=10) in the PA,
MMA, and STA, respectively. However, the maximum force developed in
response to NE (30 µmol/L) was greater in STA (1.85±0.49
mg/µm per millimeter, n=10, P<.05) compared with MMA
(0.29±0.06 mg/µmper millimeter, n=14), and PA (0.18±0.03 mg/µm
per millimeter, n=21) (Fig 1a). The
active tension produced by NE in the three types of segments, expressed
as a percentage of their own Emax (Fig 1b), was 89.8±3.5%
in STA (n=10), 34.0±5.3% in the MMA (n=14) (P<.05 versus
STA), and 20.8±3.1% in the PA (n=21) (P<.05 versus
STA).
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Figure 1. Contractile responses produced by increasing
concentrations of NE (0.01 to 30 µmol/L) in isolated human PA
(n=21), MMA (n=14), and STA (n=10). Contractions in (a) are expressed
as % of the maximum contractile responses elicited by 127 mmol/L
KCl+arginine vasopressin (1 µmol/L) and in (b) as force/unit
segment length/artery internal diameter (see "Methods"). Data are
mean±SEM. *P<.05 vs PA; +P<.05 vs
MMA.
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Responses to EFS
Voltages used to selectively activate intramural nerves
were determined using two techniques: breakthrough and subtraction (Fig 2
, for details see "Methods"). Fig 3 shows the frequency-contractile
response–curves (1 to 16 Hz) from human isolated STA, MMA, and PA to
EFS applied as square pulses of 0.3 milliseconds' duration and at
supramaximal voltage using both supramaximal and subtraction
techniques. In the STA, the contractile neurogenic responses obtained
were frequency dependent, exhibiting a threshold change at 4 Hz and
achieving 14.1±1.9% (n=21) of Emax at 16 Hz. In contrast,
in the MMA EFS-induced contractions were absent at 4 Hz and
significantly lower than those of the STA at 8 and 16 Hz. At the latter
frequency it was only 5.7±1.4% of Emax (n=5 responding
tissues of 14). In the PA, EFS contractile responses rarely occurred,
reaching 0.96±0.67% of Emax at 16 Hz (n=9 responding
tissues of 21). Unequivocal nerve-induced contractile responses were
invariably elicited from the STA segments, some of which were obtained
from the same patients whose PA segments were unresponsive.
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Figure 3. Contractile responses produced by EFS (1 to 16 Hz)
applied as square pulses of 0.3 milliseconds' duration and at
supramaximal voltage in human isolated PA (n=9), MMA (n=5), and STA
(n=21). Contractions are expressed as % of the maximum contractile
responses elicited by 127 mmol/L KCl. Data are mean±SEM.
*:P<.05 vs PA.
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Responses to ACh
ACh produced relaxation of 89.1±3.3% in the PA (n=21),
71.7±6.9% in the MMA (n=14), and 73.8±5.6% in the STA (n=10). The
large vasodilation produced by ACh in the three types of arteries
suggested that the endothelial layer was functionally
active.
Assessment of the Factors that Might Influence the Human PA
Responses to EFS
Arteries from an additional 49 patients were used to assess
possible reasons why the PA neurogenic response was absent or small
(see below). A total of 116 segments were involved. Frequently more
than one of the factors was tested in a particular segment.
Contraction to NE
The sizes of the maximum response of the PA to NE up to
3x10–5 mol/L were variable, ranging from the barely
detectable to 44% of tissue maximum (mean, 16.6±3.1). The NE
ED50 for these was 1.22±0.29x10–6 mol/L
(n=12). This was not significantly different from the value determined
in the prior series.
Patient Age and Artery Diameter
Neither the magnitude of the maximum contraction to NE, as a
percentage of Emax, nor the NE ED50 correlated
with the patient's age (r=.063; P=.076 and
r=.054; P=.84) nor with arterial
internal diameter (r=.071; P=.71 and
r=.41; P=.1, respectively).
Influence of Basal Tone
Reactivity of blood vessels to contractile agonists has been
reported to be enhanced when tone is increased.22 23 PA
autoregulate and in vivo possess intrinsic tone. In 12 segments in this
series from 5 patients, selected because they exhibited raised levels
of spontaneous tone of 16.6±1.3% Emax, the additional
contraction to NE (3x10–5 mol/L) was 20.3±3.7%
Emax. When an equivalent level of tone was induced in
segments without spontaneous tone by the addition of 17 mmol/L
K+, the NE contraction was 10.6±2.1% Emax.
These levels of response are not significantly different from the
contraction to the same dose of NE that occurred in the absence of
tone.
Responses to EFS and Reactivity to NE and ACh
Contraction
Additional experiments using EFS at 8 and 16 Hz were carried out
on arteries after equilibration in the absence of tone—fifteen using
the subtraction and four the breakthrough method of determining
stimulation voltage. Eight segments from 6 patients showed a small
TTX-sensitive contraction at 16 Hz. A response was seen in one segment
at 16 Hz, but was absent at 8 Hz. PA EFS responses were 2.74±0.55%
Emax (n=8) and 2.17±0.34% Emax (n=11) at 8
and 16 Hz, respectively. The largest response of a PA was 5.6%
Emax. The NE ED50 and NE Emax of
the pial arteries that exhibited an EFS TTX-sensitive response were not
significantly different from those that did not respond
(P=.74 and P=.63, respectively). The maximum
dilation to ACh of the segments that showed a significant TTX-sensitive
contraction was not different from those that did not
(P=.91).
Dilation
A dilator neurogenic response, if present, would be
anticipated when tone is raised. EFS at 8 and/or 16 Hz of PA that was
tonically contracted by PGF2 (3x10–6
mol/L) or that developed spontaneous tone was carried out in 10
segments. In most of these tissues some dilation, usually a small
amount, was observed on EFS, which reversed on its cessation. However,
an unequivocal TTX-sensitive relaxation was observed in only one
segment. This was 45.5% of prestimulation tone, which was about 30%
Emax. It was 13.3% after TTX. The reversible dilator
response developed very slowly to prolonged EFS.
Dilation to ACh
The mean maximum dilation to ACh of the second series was
75.8±8.4%.17 In 6 vessels relaxation completely reversed
pre-addition NE tone. The maximum ACh dilation of tissues that
exhibited EFS-induced contraction was not different from that of those
that did not respond (P=.91).
Factors that Might Mask the EFS Contractile Response
ß-Adrenoceptors
Five PA were used to test the possibility that concomitant
ß-adrenoceptor activation by an adrenergic transmitter might mask
neurogenic -adrenoceptor–mediated contraction. Four of these
arteries dilated 100%, and the fifth 46% to ACh
(3x10–6 mol/L). In three arteries, isoproterenol up
to 3x10–6 or 10–5 mol/L caused no dilation.
In one it completely reversed pre-addition tone of 24%
Emax, and in the other it caused relaxation of 43% when
the pre-addition tone level was 18% Emax. The addition of
propranolol (10–6 mol/L) did not reveal a
TTX-sensitive contraction in tissues that in its absence had not
responded to EFS, nor did it increase an existing neurogenic
contraction at 16 Hz (n=5).
L-NNA/L-NAME
Smooth muscle contraction might be diminished by concomitant
activation of the endothelium leading to release of
NO/EDRF (for example see reference 24). The NE concentration-response
relationship was determined after incubation for more than 30 minutes
in either L-NNA (10–4 mol/L) or L-NAME
(3x10–4 mol/L) when the mean increase in baseline was
3.4±1.9% Emax. NE ED50 and Emax
were not significantly altered by pretreatment with any of these agents
(P=.36 and P=.10, respectively). The contractions
to EFS (–2.6% Emax at 8 Hz) seen in 5 segments were
unaltered by exposure to these drugs.
Endothelial Factors
There was no alteration in either the size (% Emax)
or the sensitivity (ED50) of the response to NE
(P=.67 and P=.32, respectively) after
endothelium inactivation.
Indomethacin
The possibility of the concomitant production and release
of dilator prostanoids during EFS was assessed by exposure to
indomethacin (10–5 mol/L). In 3 segments
this treatment did not unmask a response to EFS where it was previously
absent. Neither the NE Emax nor the ED50 was
altered by this treatment (P=.34 and 0.26,
respectively).
Concurrent EFS Activation of Dilator Nerves
EFS during increased tone, whether spontaneous or due to
PGF2, failed to reveal a TTX-sensitive dilation either
before or after exposure to phentolamine (10–6
mol/L).
Innervation Characteristics
Catecholamine Histofluorescence
Fig 4 contains examples of the
catecholaminergic histofluorescence of PA, MMA, and
STA. Counter-staining techniques were adopted to reduce
autofluorescence. In the STA, a rich, dense, and uniform plexus
of catecholamine-containing nerve fibers could be observed.
The density of catecholamine-containing neurons was less in
the MMA. In the PA, nerve fibers were sometimes absent and when they
occurred were predominantly longitudinally oriented rather than forming
a plexus. Innervation was consistently poor compared with that
in the MMA and STA. The majority of artery segments were from the
distribution of the MCA. Pial segments from the anterior cerebral
artery distribution, although sparsely innervated, appeared
to have relatively more sympathetic neurons than those from the MCA
distribution.
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|
Figure 4. Representative photomicrographs of
catecholamine glyoxylic acid histofluorescence of
whole-mount arteries of cortical PA (550 µm ID) from a
37-year-old man (A); middle meningeal artery (750 µm ID) from a
21-year-old woman (B); and STA (1000 µm ID) from a 70-year-old
woman (C). (Magnification x400.)
|
|
Electron Microscopy
Nerve bundle density or incidence relative to the circumferential
length of the outer medial border (number/mm) of PA was assessed in 15
segments from 15 patients (see Table).
Only 13 neuronal structures were identified in a total length of
12 mm of the adventitio-medial junction (Fig 5), an incidence of approximately
1/1000 µm. The mean closest distance was 3.8±0.46 µm.
Nerve density was 26/1000 and 35/1000 µm in the MMA and STA,
respectively, and the closest nerve muscle separations were on the
order of 1 µm.
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|
Figure 5. A transmission electron micrograph of a PA from a
19-year-old woman showing a nerve bundle (NB) in the inner adventitia.
The distance to the outer medial muscle is approximately 3.1 µm.
SM indicates smooth muscle (); COL, collagen. (Bar=0.5
µm.)
|
|
|
Discussion
|
|---|
This work brings together structural and functional measurements
designed to assess the potential of the sympathetic nervous system to
induce tone in fresh human STA, MMA, and PA. Arteries were obtained
during neurosurgical procedures. The PA and MMA develop less maximum
force than the STA, and the maximum capacity to respond to NE related
to maximum force development increases in the order PA<MMA<STA. The
sensitivity of the three vessels to NE is the same. The maximum
nerve-induced responses of these three vessels were 1%, 6%, and 14%,
respectively, of their maximum contractile capacity. Our study suggests
that the basis of the marginal contractile response of the smooth
muscle of adult human PA to EFS of their intramural nerves resides in
their low density of adrenergic innervation combined with a limited
capacity of the smooth muscle cells to respond to NE. In contrast, the
capacity of the STA to contract to NE to an extent not different from
tissue maximum, combined with a more dense innervation, suggests a
significant control of this vessel by the sympathetic nervous system.
The MMA is intermediate in its reactivity.
All three types of human arteries exhibited similar sensitivities to
NE: that for the PA was 9x10–7 mol/L. Duckworth et
al10 reported an NE ED50 of
7.9x10–7 mol/L in the proximal human MCA, Shibata
9.3x10–8 mol/L,25 and Janson et al an
ED50 of 5.0±10–7 mol/L.26 Hardebo
et al27 concluded that the PA and STA were equally
sensitive to NE, with a spread of values between 10–6 and
10–5 mol/L. These values are similar to the
ED50 values reported for other human
arteries.28 29 In the rabbit, the species in which
variations in NE sensitivity and receptor affinity have been studied
the most, the ED50 of most arteries is a reasonable
approximation of receptor affinity.30 The NE dose-response
curve of the human proximal middle arteries has two components: the
first leveling at 1 x10–5 to 3x10–5 mol/L.
Higher doses are responsible for further contraction.10
This second phase is associated with the action of NE on a
"nonspecific" adrenergic receptor not influenced by
-adrenoceptor blockade, which has been called a 
-adrenoceptor or
an extraceptor, one outside the confines of the synaptic cleft. The
second component was not seen in the PA, which often relaxed to higher
doses.
In the human, as in the monkey, cat, dog, and rabbit, NE causes
contraction. However, in the cow and the pig, it elicits
relaxation.31 Another variable is the size of the NE
contraction relative to the maximum capacity to respond: that for the
PA is 20%, for the MMA and STA are 34% and 90%, respectively.
Experiments in the rabbit suggest that receptor number limits the
response of a cerebral artery.32
A semiquantitative assessment of the adrenergic innervation density was
made by catecholamine histofluorescence plus
pontamine sky blue counterstain.21 The brightness of the
fluorescence is not of quantitative significance.33
This technique has validity only when gross comparisons are made as
with the STA versus the PA. In the PA the
catecholamine-containing nerve fibers were sparse,
contrasting with the much denser uniform plexus of aminergic nerve
fibers observed in the STA. There were intermediate levels in the
MMA.
Perivascular catecholaminergic nerves have been identified
by specific histofluorescence in a number of species, for
example, the cat and monkey pial arteries34 35 and
rabbit12 and human10 36 middle cerebral
arteries. The latter authors describe a fairly thick, densely packed,
mesh-like system of varicose sympathetic fibers in the adventitia and
outer media of the STA and PA. They did not distinguish between the two
types of arteries. They found a broad multiaxonal plexus, typical of
preterminal axons in the outer adventitial layer. No bright focal
collections indicative of terminal axons were seen.
These general conclusions are confirmed and extended by quantitative
electron microscopy. In the STA the nerve bundle density was
35/1000 µm. In the PA, it was 1/1000 µm. Between these
two extremes is the middle meningeal artery with 25 bundles/1000
µm. In the PA, the few nerve bundles seen were widely separated from
the closest smooth muscle cells.
Two technical approaches to the determination of the stimulation
voltage for the EFS of the perivascular nerves were used. Both used
TTX. It is assumed that the TTX-sensitive component of the change in
force with EFS reflects action potential–initiated transmitter
release. The pulse duration adopted (0.3 milliseconds) provides optimum
selectivity for nerve activation in the intact tissue.1 In
some experiments a pulse duration of 0.6 milliseconds failed to
influence the size of the response. A frequency of 8 Hz, and sometimes
16 Hz, is known to provide maximum or near maximum neurogenic
responses. Tissues were exposed to NE before EFS in an attempt to
replenish neuronal transmitter stores. In the breakthrough technique,
the maximum voltage that just failed to activate smooth muscle
cells was used. The main criticism of this method is that the voltage
used may not activate all the perivascular nerves. With the
subtraction method, the responses to pulses delivered at voltages set
above the threshold for muscle activation are reduced by those
responses seen after nerve inactivation with TTX. One criticism of this
approach is that if there is concomitant muscle depolarization caused
by the EFS, vascular smooth muscle responsiveness to released
neurotransmitter could be altered. The level of the membrane potential
is an important determinant of smooth muscle reactivity. The comparable
findings obtained by both approaches increases our confidence in the
conclusions.
In no instance was an unequivocal nerve response greater than 5.6% of
the maximum possible contraction of the human PA observed. Among those
that contracted, it varied from threshold to 3%. No neurogenic
response was seen in 60% of all the artery segments studied. This is
in agreement with the observations of Hardebo et
al.12 Fresh human arteries were examined both in
the study by Hardebo et al and in the current study. In postmortem
human MCA,10 neurogenic responses were absent in 80% of
tissues that were examined within 4 hours after death. Mean responses,
when they occurred, were about 6%. In autopsy material Shibata et
al8 recorded that 2 of the 4 segments of human MCA
(diameters unspecified) from patients aged 63 to 68 years exhibited
neurogenic responses that were between 25% and 30% of NE
Emax. They used extreme parameters of
stimulation: 80 V, 100 Hz, and duration 10 milliseconds. The
contraction was TTX- and phentolamine-sensitive. Toda and
Fujita11 were unable to demonstrate nerve responses in
basilar, posterior cerebral, and intracranial carotid systems in
vessels obtained after death. Sizable neurogenic responses, both
constrictor and dilator, have been found by many laboratories in major
cerebral arteries of a variety of animal species, for example, the
rabbit,37 dog and sheep,38 cat,39
and monkey.35 To generalize, human proximal cerebral
arteries appear to be modestly responsive to nerve activation. PA are
virtually unresponsive. The STA was the most responsive to EFS and the
MMA was intermediate. The STA is a cutaneous artery. The cutaneous
vessel whose adrenergic control has been analyzed in some
detail40 is the rabbit central ear artery. Neurogenic
responses of the main trunk and the first- and second-order branches of
that artery are 73%, 60%, and 35%, respectively, of maximum. This
artery, however, probably has the highest density of innervation of any
artery studied.
Some additional factors that might contribute to the poor sympathetic
response were assessed. The possibility was considered that the poor
neurogenic response might have resulted from the overnight storage.
However, these conditions did not influence the neurogenic constriction
of the rabbit ear artery7 or that of the human STA. The
neurogenic responses of the PA that did occur were not restricted to
first-day studies. The tissue samples did not come from a predominantly
aged population. The NE response did not diminish with age between 15
and 75 years, a feature observed in some vascular
preparations.15 A sizable nerve response was rarely found
in any tissue. The sensitivity of the -adrenoceptor to NE in the PA
is not uniquely low. Furthermore, responses did not change when NE was
added to a vessel with background tone. Finally, EFS would potentially
activate all nerves in the artery wall, both constrictor and
dilator. However, when tone occurred spontaneously or was induced with
an agonist, EFS failed to reveal a TTX-sensitive dilation; nor was this
observed in the main MCA under similar circumstances after
-adrenoceptor block with phentolamine.10
Nerve-induced dilation of PA has not been recorded in any animal
species (see for example reference 12), although this is the dominant
response in the proximal cerebral arteries of the cat39 and
pig.41 A variety of dilator nerves have been visualized
using different techniques in a number of species42 43 44 :
those containing ACh,42 43 neuropeptide Y, vasoactive
intestinal peptide, substance P, calcitonin gene-related
peptide,43 44 45 and NO.46 Evidence for a
functional role for some of these dilator neurotransmitters in nonhuman
arteries has been reviewed.47 Our electron microscopy
survey does not identify specific types of neurons in the artery wall,
and we can only conclude that innervation of any form is present
only in minimal amounts.
There is little evidence for a significant ß-adrenoceptor population
in cerebral arteries.48 49 The inconsistent dilator
effect of isoproterenol in the present studies suggests that
ß-adrenoceptors could not account for the consistent absence
of a neurogenic contractile response. Furthermore,
propranolol failed to reveal neurogenic dilation where this
was previously absent or to potentiate a response when manifest.
EDRF/NO influences basal and constrictor tone in the cerebral
circulation.25 Exposure to L-NNA or L-NAME, and also
endothelium removal, had no significant effect on the
response of the PA to NE or EFS. Local endogenous
production of dilator prostanoids can modulate vascular
responsiveness.50 Treatment with
indomethacin (10 µmol/L) did not change
consistently reactivity of the PA to NE or EFS.
This study supports the conclusion that in the human PA the marginal
neurogenic contractile response reflects innervation of low density
separated widely from smooth muscle cells. Compounding this picture is
the low capacity of the artery to respond to NE. By contrast, the STA
has a sizable neurogenic response, because it has a high nerve density;
the nerve bundles are closer to the smooth muscle cells and respond
well to NE. Since PA in toto comprise an important part of an effective
autoregulating bed and since the role of the sympathetic nerves is
questionable, pressure and flow are undoubtedly major regulatory
mechanisms. The sympathetic nervous system may have roles other than
those assessed in this study. It has been suggested that the
innervation extends the range of autoregulation in vivo.2
Sympathetic innervation has been claimed to be protective against
stroke and hypertension in animals,51 52 possibly
reflecting a neurotrophic role,53 particularly during
development.
|
Selected Abbreviations and Acronyms
|
|---|
| ACh |
= |
acetylcholine |
| EFS |
= |
electrical field stimulation |
| L-NAME |
= |
NG-nitro-L-arginine methyl ester |
| L-NNA |
= |
NG-monomethyl-L-arginine |
| MCA |
= |
middle cerebral artery(ies) |
| MMA |
= |
middle meningeal artery(ies) |
| NE |
= |
norepinephrine |
| PA |
= |
pial artery(ies) |
| PGF2 |
= |
prostaglandin F2 |
| STA |
= |
superficial temporal artery(ies) |
| TTX |
= |
tetrodotoxin |
|
|
|
Acknowledgments
|
|---|
This study was supported by the Totman Medical Research
Fund.
|
Footnotes
|
|---|
Some of this material has been reported previously in Human Brain Circulation, Chapter 7, Rosemary D. Bevan, John A. Bevan, eds. Totowa, NJ: Humana Press; 1994.
Received June 23, 1997;
revision received October 15, 1997;
accepted October 15, 1997.
|
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Editorial Comment
Tony J-F. Lee, PhD
Department
of Pharmacology,
Southern Illinois University School of Medicine,
Springfield, Illinois
|
Introduction
|
|---|
The functional significance of sympathetic innervation in
regulating vascular tone is dependent on multiple factors, including
density of innervation, synaptic cleft distance, receptor type,
receptor population and characteristics, and coupling mechanisms. These
factors are markedly variable among species and from region to region
within the same species. In the cerebral circulation of animal models,
dense sympathetic innervation is found in arteries at the base of the
brain. The density of the innervation decreases gradually with a
decrease in arterial diameter and disappears in the small pial
arteries. Accordingly, the pial arteries are very weak or unresponsive
upon stimulation of sympathetic nerves. On the other hand, pial veins
from some species, such as the pig, receive denser sympathetic
innervation1 and are more sensitive to norepinephrine (NE)
than pial arteries.
In the study reported here, Bevan and colleagues demonstrated that
freshly obtained human pial arteries, like those of experimental
animals, receive sparse adrenergic/sympathetic innervation. These
authors further demonstrated that, compared with human middle meningeal
and superficial temporal arteries, human pial arteries were only
marginally responsive to transmural nerve stimulation and exogenous NE.
Bevan et al also examined the mechanisms responsible for weak
reactivity to adrenergic/sympathetic nerve stimulation. Pial artery
responses to transmural nerve stimulation were not affected by
propranolol, inhibitors of nitric oxide synthase and cyclooxygenase, or
endothelial denudation. Results from histochemical and ultrastructural
studies demonstrated that pial adrenergic nerve density was sparse, and
synaptic cleft distances were largely compared with middle meningeal
and superfucial temporal arteries. Results of this structural and
functional correlation study suggest that the weak neurogenic response
of adult human pial arteries reflects their poor innervation and
responsiveness to NE, implying that these features are not important in
the regulation of the arteries' diameters. This study extends a
variety of findings on sympathetic control of pial arterial tone made
in experimental animals to the human cerebral circulation.
The conclusion given by the authors on the functional significance of
pial artery sympathetic nerves is logical. These authors imply that NE
released from the sympathetic nerves is ineffective in directly causing
constriction of the smooth muscle. Recently, it has been shown that NE
released from the adrenergic nerves can elicit release of nitric oxide
from the neighboring nitric oxidergic nerves,2 suggesting
that transmitter NE can indirectly affect the pial arterial tone. This
possibility, however, cannot be concluded from results of the Bevan et
al study in human pial arteries since the presence of nitric oxidergic
or other autonomic innervation in these arteries was not evaluated. The
exact functional significance of the pial artery sympathetic
innervation in humans remains to be determined. Since isolated pial
arteries were used in this study, the results of these findings may be
more reasonable in suggesting that the perivascular sympathetic
innervation in pial arteries is not important in direct regulation of
its diameter.
|
Selected Abbreviations and Acronyms
|
|---|
| ACh |
= |
acetylcholine |
| EFS |
= |
electrical field stimulation |
| L-NAME |
= |
NG-nitro-L-arginine methyl ester |
| L-NNA |
= |
NG-monomethyl-L-arginine |
| MCA |
= |
middle cerebral artery(ies) |
| MMA |
= |
middle meningeal artery(ies) |
| NE |
= |
norepinephrine |
| PA |
= |
pial artery(ies) |
| PGF2 |
= |
prostaglandin F2 |
| STA |
= |
superficial temporal artery(ies) |
| TTX |
= |
tetrodotoxin |
|
|
Received June 23, 1997;
revision received October 15, 1997;
accepted October 15, 1997.
|
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Top
Abstract
Introduction