316
Aminergic Histofluorescence and Contractile
Responses to Transmural Electrical Field
Stimulation and Norepinephrine of Human
Middle Cerebral Arteries Obtained
Promptly After Death
John W. Duckworth, George C. Wellman, Carrie L. Walters, and John A. Bevan
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
The responses of cerebral arteries to catecholamines and sympathetic nerve stimulation show
wide variation between animal species. We examined the catecholaminergic histoflnorescence
and the contractile responses elicited by transmural electrical field stimulation and norepinephrine (NE) in proximal segments of human middle cerebral artery (MCA) obtained during
autopsy. Twenty-four percent of the specimens were obtained within 2 hours and 76% within
4 hours of death. A moderately dense catecholaminergic histofluorescence was seen in all
segments of human MCA using the gfyoxylic acid technique, counterstained with pontamine sky
blue. However, only seven of 35 (20%) MCA segments tested showed tetrodotoxin-blocked
transmural electrical Seld stimulation contractions, and all of these were harvested within A
hours of death. The responses were mostly seen in the most proximal MCA segments and, at 32
Hz, only achieved 6 ± 1 % of the maximal tissue contraction. NE caused two distinct responses
in human MCA segments. At low concentrations, It acts via an o-like adrenoreceptor to cause
contractions 20 ± 3 % of the maximal tissue response. The NE ED^s for the three successive
segments were not different from each other; the value for the most-proximal segment was
7.9±0.2xl0" 7 M. At concentrations above 10"s M, this catecholamine acts on low-affinity sites
resistant to a-adrenergic antagonists causing contractions that at 10~3 M reach 52 ±5% of the
maximal tissue response. Our results suggest that it is important when studying human blood
vessels to harvest them as soon as possible after death, that the smooth muscle response to
sympathetic activation is small, frequently absent, and that the postsynaptic sympathetic
mechanism includes not only o-adrenoceptors but low-affinity sites as well. (Circulation
Research 1989;65:316-324)
A
dense, catecholaminergic innervation within
the adventitia of cerebral arteries has been
>. consistently identified using a histofluorescent technique in many species, including man. 1 - 5
However, there seems to be a considerable species
variation in the type and subtype of adrenoreceptors on the vascular smooth muscle cells in cerebral
arteries.* For example, segments of rabbit large
cecetJEji arteries show responses mediated by ar
like adrenoreceptors while responses of equivalent
From the FJmsioft of Neurological Surgery, Department of
Swgefy, the Department of Pharmacology, University of Vermont, Burlington.
Supported by US Public Health Service Grant HL-32383.
Address for reprints: John A. Bevan, MD, Department of
Pharmacology, University of Vermont, Given Medical Building,
Budwgten, VT 03405.
Received July 20, 1988; accepted January 13, 1989.
segments from the adult pig depend exclusively on
/}-adrenoceptors. 7 - 9 The norepinephrine (NE)induced contractions of dog cerebral arteries are
blocked by yohimbine but not by prazosin, suggesting the presence of a2-adrenoreceptors, while the
receptors in the monkey and human, like the rabbit,
exhibit a ^ adrenoreceptor-like properties.10 Thus,
generalization cannot be made from single animal
studies, and extrapolation to the human is unreliable.
Demonstrations of catecholamine innervation by
histofluorescence in human cerebral arteries are
scant,4-3'11 and the adrenergic responses of these
arteries are mentioned in only a few reports. Early
studies simply showed the relative insensitivity of
human cerebral compared with systemic arteries to
exogenous NE. 1 2 - 1 4 Later investigations surveyed
human cerebrovascular responses to a number of
pharmacological agents,15-16 but often the same iden-
Duckworth et al Human Middle Cerebral Artery
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
tified vascular segment was not used in the experimental series, and arteries were usually obtained
8-60 hours after death.
The present study was undertaken to evaluate
some of the catecholaminergic mechanisms present
in three standardized segments of human proximal
middle cerebral arteries (MCAs) harvested never
more than 6 hours, and in the majority of instances
much more promptly, after death. Catecholamine
histofluorescence and the contractile response of
isolated segments in vitro to transmural electrical
field stimulation (TEFS) and exogenous NE were
investigated.
Only 20% of the MCA segments exhibited neuralmediated contractions, and these were small. The
NE responses were the composite of small responses
initiated by lower doses via a-adrenoreceptors and
of larger contractions originating from low-affinity
sites not influenced by a-adrenoceptor antagonists.
These results provide quantitative information
regarding the adrenergic neuroeffector mechanism
of human MCAs obtained under as ideal conditions
as practicable, allowing the artery to be placed in
the perspective of similar studies on cerebral arteries of other species. The human adrenergic neural
vascular mechanism has recently been reviewed.17
A preliminary communication of this paper has
been previously made.18
Materials and Methods
When autopsy permission had been obtained after
a hospital death, the brain was removed as soon as
possible using standard autopsy procedures.19 The
MCA was dissected free of surrounding arachnoid
from its origin at the internal carotid to beyond a
tertiary branching point as it coursed through the
Sylvian fissure. Care was taken in handling the
vascular segment during removal. It was immediately placed in Krebs' solution (millimolar concentration: Na + 144.2, K+ 4.9, Ca2+ 1.3, Mg2* 1.2, CT
126.7, HC0 3 - 25.0, SCV 1.19, glucose 11.1, CaEDTA 0.023, ascorbic acid 0.11) at room temperature gassed with 95% O2-5% CO2, maintaining pH at
7.4 for transport to the laboratory.
Three-millimeter segments of MCA were obtained:
1) from the most proximal segment (Ml), 2) from
just past its first branch (M2), and 3) from just past
the second branching point (M3). Two segments
were taken from each site. Only segments free of
frank atherosclerosis and without any adherent blood
clots were studied.
One segment from each area was mounted for in
vitro study of isometric contraction in a doublejacketed tissue bath.20 Ring segments were cannulated with two 30-gauge wires under a dissecting
microscope. One wire was bent into a " U " shape to
fix the segment to a stationary bar in the bath. The
other wire was attached to a tension transducer
(model FT03, Grass Instruments, Quincy, Massachusetts) mounted on a base moved by a micrometer. This allowed the segment to be stretched.
317
Changes in isometric force were recorded on a
Soltec model 220 strip chart recorder (Soltec Corp.,
Sun Valley, California). Platinum wire electrodes
(0.3 mm diameter, 3 mm long) were placed on either
side of the suspended vascular segments. The electrodes were connected to a Grass stimulator and
subsequently used for transmural electrical field
stimulation. Each vascular segment was equilibrated for 90 minutes in Krebs' solution maintained
at 37° C and gassed continuously with 95% O2-5%
CO2. The bathing solution was changed every 15
minutes. Vessels were then stretched in a step-wise
manner to optimum preload of 2.0 g for Ml, 1.5 g
for M2, and 1 g for M3 segments. These preloads
were determined from active tension-length curves
in preliminary experiments for this project.
After 30 minutes of equilibration at a stable
preload in which Krebs' solution was replaced
twice, a trial period of TEFS was carried out in the
presence of propranolol ( 1 0 s M) and atropine
(10"s M) to eliminate possible ^-adrenergic or cholinergic effects. Fifteen-second trains of squarewave pulses of 0.3-msec duration were delivered to
the electrodes at 16 Hz. At 10-minute intervals,
stimulation was made at 4, 8, 10,15, and 20 V. If no
constrictor response was recorded, TEFS trains of
2 minutes' duration using the same pulse parameters with step-wise increases in voltage were applied.
If again no response was seen, the segments were
tested for responses to exogenously applied NE. If
segments contracted to TEFS, tetrodotoxin (TTX;
10"7 M) was added to the bathing solution, and the
stimulation sequence was repeated 30 minutes later
until contraction was seen. The protocol was
designed to find the highest TEFS voltage causing a
contractile response that was completely eliminated
by adding TTX. If all TEFS responses to the initial
voltage series were completely eliminated by TTX,
then a new stimulation trial was carried out that
involved higher stimulation voltages. After washing
the segments (Krebs' solution changed every 15
minutes three times), the highest TTX-blocked TEFS
voltage was applied at different frequencies (2, 4, 8,
16, 20, and 32 Hz) in a random sequence. From this,
the frequency-response relation for TEFS was
obtained with stimulation parameters that were
completely eliminated by TTX. If contractions were
reduced but not completely eliminated by TTX at
all voltages eliciting a contraction, the segments
were washed as above, and the TEFS sequence of
increasing voltage was repeated. Subtraction of
these responses was considered the TTX-sensitive
component of contraction, presumably that associated with neural stimulation.
Contractions to the cumulative addition of NE
(10~8—10~3 M) were recorded from all segments 15
minutes after the addition of propranolol (10~6 M)
and deoxycorticosterone acetate (10~6 M) to eliminate /3-adrenoreceptor and uptake2 (extraneuronal)
mechanisms during dose-response measurements.
We were unable to use desmethylimipramine
318
Circulation Research
Vol 65, No 2, August 1989
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
(10 7 M) to inhibit uptake! (neuronal) since it caused
a significant reduction rather than the anticipated
increase in the NE response. This probably represented the a-adrenoceptor antagonist properties of
this agent. In three cases, NE dose-response
sequences were repeated in the presence of phentolamine (10~6 M). In another three instances, doseresponse sequences were repeated after exposure
to phenoxybenzamine 3xl(T 8 M for 15 minutes
followed by washing for 1 hour. After the NE
dose-response curve sequence was complete, the
arteries were washed, and the maximal contraction
of the segment (tissue maximum) was elicited by
cumulative addition of NE (10~3), prostaglandin F ^
(1CT5 M), serotonin (10~4 M), and potassium chloride (100 mM) to the bath.
The other MCA segments obtained were prepared for examination of catecholaminergic histofluorescence of perivascular nerves. Segments processed by the glyoxylic acid method21 showed a
sporadic, moderately dense, perivascular fluorescent plexus characteristic of catecholaminergiccontaining nerves. However, the intensity of fluorescence was low and not easily distinguished from
background autofluorescence. Neuronal fluorescence was intensified by incubating vascular segments in NE (10"2 M) and pargyline (10~6 M) in
Krebs' solution for 20 minutes and then exposing
them to pontamine sky blue (0.5% wt/vol) and
glyoxylic acid (2% wt/vol) mixed in phosphate buffer
(0.1 M; pH adjusted to 7.0 with ION sodium hydroxide, notated NaOH) for 30 minutes at room temperature. Segments were then rinsed twice with phosphate buffer, and the circular segments were cut
open and spread on glass slides with the endothelial
surface down. Slides were air dried and then placed
in a convection oven at 100° C for 4 minutes.
Tissues were immediately covered with oil. Observations of histofluorescence were made as soon as
possible with a Nikon fluorescent microscope.Medical records were reviewed on all cases.
Special attention was given to age, sex, medications, illnesses ^past and present), blood gasses,
and serum chemistries as indicators of the "viability" of vascular segments obtained and the
reason for any variation that may be observed
during the experimental protocol.
Statistics
All results are reported as mean±SEM. Significance was accepted using the Student's t test,
when/><G.O5.
Results
Hooam MCAs were o*rtamed from 21 autopsies
less than 6 hours after death. Twenty-four percent
were harvested less than 2 hours afteT death, and
76% were oblaifted less than 4 hours after death.
The iH*mediate causes of death are listed in Table 1.
Ages ranged from 27 to 90 years; the average age
was 64. No specknees were accepted from patients
TABLE 1. Immediate Came of Death of Donors of Cerebral
Arteries and the Incidence of Their Adequate In Vitro Response to
Norepinephrine and Tetrodotoxln-Seiisitrve Tmnsmural Electrical
Field Stimulation
Sudden death
(MI, arrhythmia)
Sepsis
Brain death
Exsanguination
Respiratory
insufficiency
Unknown
Totals
Cases
NE
TEFS
6
6
3
3
2
4
1
1
2
2
0
2
1
0
0
1
0
20
8
5
0
NE, adequate norepinephrine dose-response curve obtained;
TEFS, tetrodotoxin-sensitive response to electrical field stimulation; MI, myocardial infarction.
who had a clinical history of diabetes, dysautonomia, or other diseases that might involve the efferent
postganglionic sympathetic neurone, or who had
had prolonged drug therapy considered likely to
affect specifically the sympathetic-related responses
that were the object of this study.
Not all segments contracted to the agonists
employed. Despite rapid vessel recovery, four of 21
(19%) cases showed no contractions. However,
three of these were obtained 5-6 hours after death.
The fourth group of segments were from a 69yeai-old woman who died after prolonged respiratory insufficiency.
All other vascular segments exhibited contractile
responses, but adequate data allowing quantitation
of responses to TEFS and NE cumulative dose
responses could not be obtained in six segments
(29%). Spontaneous NE-induced oscillating or rhythmic contractions occurred in four instances (see
Figures 1A and IB), precluding a quantitative study.
In two other instances, arteries did not constrict to
TEFS or NE but did respond to prostaglandin F ^ .
From eight cases, vascular segments that showed
graded maintained contractions when exposed to
cumulative NE concentrations were obtained. Five
cases produced vascular sepaents that showed' "TTXsensitive" TEFS contractions (see Table 1). All
cases showing NE or TTX-sensitive TEFS contractions were obtained less than 4 hours after death.
Catecholamine Histofluorescence
The histofluorescence of catecholaminergic nerves
was difficult to demonstrate in human cerebral arteries using the standard glyoxylic acid technique.21
After coixnterstajning with pontamine sky blue,22 a
broad ffitritiaxoaal plexus, typical of preterminaJ
axons, was seen in the outer adventitial layer.
However, no bright focal collections usually indicative of terminal axons were seen (Figure 2, top).
Although quantitative assessment was not made,
there was no obvious difference in the appearance
or magnitude of this fluorescence between Ml, M2,
Duckworth et al
Human Middle Cerebral Artery
319
HUMAN MIDDLE CEREBRAL ARTERY
lOmin
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
FIGURE 1. Part of experimental traces
of segments of human middle cerebral
arteries showing spontaneous rhythmic
activity. A: Spontaneous and B: after
addition of norepinephrine (10~7 M).
e
e
O
IT
O
10 min
NorepmephnnedO
and M3 segments. When segments were incubated
with NE (10"2 M) and pargyline (1(T2 M),finebright
fluorescent axonal bundles typical of terminal systems were observed (Figure 2, bottom). These
terminal varicosities were best seen in smaller,
thinner segments of M3 but were also present in M2
and Ml segments. On the basis of absence of its
characteristic fluorescence, no serotonin-containing
neurons were observed.
Transmural Electrical Field Stimulation
TTX-sensitive contractions could be elicited by
TEES in seven arterial segments from five cases
(see Table 2). In three segments, stimulation parameters were found that caused contractions that were
completely eliminated by TTX (10~7 M) and that
returned after washing. In two segments, we could
only determine stimulation parameters that produced contractions that were reduced by TTX
(10~7 M) but that recovered after washing. In eight
cases, TEFS contractions were not reduced by
TTX. These were typically seen with high voltages
at 16 Hz. The number of Ml segments exhibiting
TTX-sensitive TEFS was greater than the number
of M2 or M3 segments showing this response
(p<0.01). These TTX-sensitive contractions were
seen in 50% of the Ml segments but only in 8% of
the M2 and 12% of the M3 segments (Table 2).
Segments exhibiting TTX-sensitive contractions
tended to be from younger patients, although this
conclusion did not reach statistical significance.
The TTX-sensitive group was not different from the
TTX-insensitive group with respect to illness or
time after death although the series is probably too
small to allow a conclusion. To quantitate the
"neural" contraction, we pooled the greatest contraction that was subsequently shown to be TTXsensitive elicited during the standard experimental
protocol, when expressed as a percent of the maximal tissue response, for all responding segments
(Figure 3). The parameters used to elicit these
contractions were generally 10 V at 32 Hz. The
mean TTX-sensitive component of TEFS contraction was 6.0±1.3% (H=7) of the tissue maximum
contraction.
In a majority of the segments, TEFS was also
applied when tone was raised. Using a voltage
previously shown to be just short of breakthrough
for contractions in the presence of tetrodotoxin
(see "Materials and Methods"), no relaxation was
ever observed.
322
Circulation Research Vol 65, No 2, August 1989
HUMAN MIDDLE CEREBRAL ARTERY
e
NOftEPNEPHRINE (-
j
NCREPlNEPHFMNE
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
X 60-1
.NOREPMEPHFIINE 1-loqM)
NOREPINEPHRNE (-logMl
4. Norepinephrine (NE) dose-response curves from human middle cerebral artery. Plotted as percent of the
maximal NE contraction (A), percent of the maximal contraction for the segment (B), percent of the contraction to
10"* M NE (C), and percent of the maximal NE contraction in M2 segment in the presence ofphentolamine 10'6 M (D).
O, most-proximal segment (Mi); •, segment just past the first branch (Mj); A, segment from just past the second
branching point (AfJ. Number of arterial segments studied: M,, five; M^ eight; M3, five (A, B, and C, respectively); M*
three (D).
FIGURE
In contrast to some other human arteries,17
attempts to activate vascular smooth muscle cells of
the human cerebral artery by electrical stimulation
of their perivascular neural elements has proven to
be difficult. There are two early reports of successful neuronal stimulation leading to contraction. Shibata et al27 found TEFS contractions blocked by
TTX or phentolamine in two of four MCA segments
tested. However, their stimulation parameters were
extremely high (80 V, 100 Hz), pulse duration was
long (10 msec), and only very small phasic contractions were produced. Edvinsson et al11 reported
TEFS contractions of human adult pial arteries but
show only one frequency-response curve. These
authors do not describe this result in detail, and
their rate of success and consistency of responses
was not mentioned. Others have reported an inability to eticit TEFS contractiofl in humaa arteries,
both from the circle of Willis2816 and from the pial
surface.28 We find the TEFS to be inconsistent, and
when it does occur, it is less than 10% of the
maximal contraction for the arterial segment. This
seemingly poor response is not the consequence of
the experimental set-up. We have been able to
obtain consistently a neural-mediated TEFS contraction in human superficial temporal arteries
removed at surgery that were 26% of the maximal
NE response for the segment (J. Duckworth, unpublished data). Furthermore, the same experimental
procedure has provided consistent responses in
many different arteries from many species (for
example, see Bevan and Bevan29 and Lee et al30).
The lack of TEFS response may not necessarily be
true of all cerebral arteries and may reflect a regional
variation in the cerebral arteries. Bevan and Bevan29
found that only the proximal rabbit basilar artery
responded to TEFS. Likewise, we found a TTXsensitive component of TEFS in the proximal MCA
(i.e., Ml) much more prominent than in more distal
branches of the same artery. The overall weak
effects of neural-mediated contraction may be due
to postmortem change but may also reflect the high
threshold of these arteries to NE associated with
the low affinity of the a-adrenoceptors, the relative
low density of these receptors, the presence of
low-affinity sites that mediate NE contraction, and
the wide adrenergic synapse that has been reported
for cerebral arteries, at least from the cat.
Duckworth et al Human Middle Cerebral Artery
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
The electrically mediated contraction of animal
cerebral vessels shows other unusual characteristics. Lee et al30 found that TEFS contractions of
rabbit basilar artery were increased after aadrenoceptor blockade with phenoxybenzamine and
phentolamine. Araki et al33 found no denervation
supersensitivity of rabbit or cat cerebral arteries,
even though aminergic histofluorescence and TEFS
contractions were eliminated by sympathectomy. A
generally acceptable explanation of these features
has not yet been put forward.
The NE-mediated constriction of human cerebral
arteries also shows some unusual features. On a par
with the contractions to TEFS, NE causes only a
weak response of human cerebral vessels.11-13 In
this study, only 20% of the tissue maximum is
reached when 10~4 M NE is present. This is in good
agreement with other reports.11-13 At 10~5 M NE,
Rose and Moulds15 found that large human cerebral
vessels contracted 27.42% of that elicited by potassium chloride (80 mM). In this regard, they differed
significantly from human digital arteries when this
value was 136.77%. The contraction of human cerebral arteries to less than 10"4 M NE is mediated
through an a-like adrenoreceptor since it is eliminated by phentolamine and also phenoxybenzamine, a result similar to that found in the rabbit
basilar artery.34
Additional development of force occurred when
NE exceeded 10~4 M. In the presence of 10' 3 M
NE, 52% of the tissue maximum was reached, and
this additional contraction was both phentolamine
and phenoxybenzamine resistant. This effect has
been previously demonstrated in cerebrovascular
studies of animals, particularly the rabbit,34 but not
man. In the rabbit, such contractions occur with a
variety of adrenoreceptor agonists35 and, at 10"3 M
NE, are approximately 60% of the maximal tissue
contraction for the segment.7 They are not blocked
by a-adrenoceptor, histamine, or serotonin antagonists. 31 - 33 - 34 They may be similar to the yadrenoceptor reported by Hirst and Neild.36-38 Such
sites of drug action have been given the alternative
name of extraceptors. The role of these receptors,
if any, in adrenergic transmission is problematical.- Postsynaptic NE concentrations may be
in excess of 10"3 M33-42 and would be sufficient to
activate such sites, but whether this would result in
effective transmission is not known.
The EDW for the first part of the MCA NE
dose-response curve, which up to 10~4 M is blocked
by phentolamine and phenoxybenzamine, is
lxlO" 5 M. This is similar to published data for the
human basilar27 and MCA segments.43 Other reports
are quite different and range from 9.8 xlO" 9 M12-27
to 2.05XlO"7 M.10 These discrepancies may be due
to several factors. First, previous studies of human
cerebral arteries did not always use an exactly
identified segment,11'12"15'44 and regional variation
within the same vessel can occur. Secondly, previous studies have used arterial segments obtained
323
long after death (i.e., greater than 8 hours).13-15
Toda et al45 has suggested that relative NE activity
in human cerebral arteries remains intact up to 20
hours after death, but these authors do not detail
the NE dose-response curve. Thirdly, records of
human cerebral vascular tissue are often difficult to
analyze. Spontaneous oscillating contractions like
those shown in Figure 1 have plagued researchers, ii.i4.44 and pronounced interindividual and intraindividual variation of human cerebral artery segments has been found.15-44'46 When the same arterial
segment is analyzed as soon after death as possible,
these difficulties may be minimized, since, in our
study, variation in sensitivity was small.
Finally, another important factor that complicates
the measurement of catecholaminergic sensitivity is
the presence of both normal and low-affinity sites.
NE EDso is obviously influenced by the choice of the
response that is considered maximum. We suggest
that NE acts in human and animal vascular tissue at
two sites: 1) a low-dose, moderate-affinity site inhibited by adrenoceptor antagonists and 2) a high-dose,
low-affinity site insensitive to these drugs. NE ED^
has significance only when one response component
is measured at a time. The limitation of the size of the
first phase, to some 20% of the maximum tissue
response, may reflect a low receptor number; at least
this seems to be the case for the rabbit.31-39
Acknowledgment
It is a pleasure to thank Dr. N. Harding for his
helpful cooperation, assistance, and discussions.
References
1. Nielsen KC, Owman C: Adrenergic innervation of pial
2.
3.
4.
5.
6.
7.
8.
9.
arteries related to the Circle of Willis in the cat. Brain Res
1967;6:773-776
Peerless SJ, Yasargil MG: Adrenergic innervation of the
cerebral blood vessels in the rabbit. / Neurosurg 1971;
35:148-154
Hernandez-Perez MJ, Stone HL: Sympathetic innervation
of the Circle of Willis in the Macaque monkey. Brain Res
1974;80:507-511
Barrett RE, Fraser RAR, Stein BM: A fluorescence histochemistry survey of monoaminergic innervation of cerebral blood vessels in primates and humans. Trans Am Neurol
Assoc 1971;96:39-45
Akiguchi I, Fukuyama H, Kameyama M, Koyama T, Kimura
H, Maeda T: Sympathetic nerve terminals in tunica media of
human superficial temporal and middle cerebral arteries:
Wet histofluorescence. Stroke 1983;14:62-66
Bevan JA: Autonomic pharmacologist's guide to the cerebral circulation. Trends Pharmacol Sci 1984^:234-236
Laher I, Khayal MA, Bevan JA: Norepinephrine-sensitive,
phenoxybenzamine-resistant receptor sites associated with
contractions in rabbit arterial but not venous smooth muscle:
Possible role in adrenergic neurotransmission. J Pharmacol
Exp Ther 1986;237:364-368
Lee TJ-F, Kinkead LR, Sarwinski S: Norepinephrine and
acetylcholine transmitter mechanisms in large cerebral arteries of the pig. J Cereb Blood Flow Metab 1982;2:439-450
Winquist RJ, Webb C, Bohr D: Relaxation to transmural
nerve stimulation and exogenousry added norepinephrine in
porcine cerebral vessels: A study utilizing cerebrovascular
intrinsic tone. Ore Res 1982;51:769-776
324
Circulation Research
Vol 65, No 2, August 1989
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
10. Toda N: Alpha adrenergic receptor subtypes in human,
monkey and dog cerebral arteries. / Pharmacol Exp Ther
1983;226:861-868
11. Edvinsson L, Owman C, Sjoberg N-O: Autonomic nerves,
mast cells and amine receptors in human brain vessels. A
histochemical and pharmacological study. Brain Res 1976;
115:377-393
12. Allen GS: Cerebral arterial spasm. Part 5: In vitro contractile
activity of vasoactive agents including human CSF on human
basilar and anterior cerebral arteries. J Neurosurg 1976;
44:594-600
13. Starling LM, Boullin DJ, Grahame-Smith DG, Adams CBT,
Gye RS: Responses of isolated human basilar arteries to
5-hydroxytryptamine, noradrenaline, serum, platelets and
erythrocytes. J Neurol Neurosurg Psychiatry 1975;
38:650-656
14. Miller CA: Biochemistry of vascular smooth muscle: Contractile mechanism of human basilar artery, in Wilkins RH
(ed): Cerebral Arterial Spasm. Baltimore, Williams & Wilkins Press, Chapter 10, 1980, pp 68-75
15. Rose GA, Moulds RFW: Pharmacological comparison of
isolated human cerebral and digital arteries. Stroke
1979;10:736-741
16. Toda N, Fujita Y: Responsiveness of isolated cerebral and
peripheral arteries to serotonin, norepinephrine and transmural electrical stimulation. Ore Res 1973;33:98-104
17. Bevan JA: The human adrenergic neurovascular mechanism. Gen Pharmacol 1983;14:21-26
18. Duckworth J, Walters C, Bevan JA: Unique adrenergic
properties of human middle cerebral arteries (abstract).
Stroke 1988;19:129
19. Baker RD: Postmortem examination, in Specific Methods
and Procedures. Philadelphia, WB Saunders, 1967, pp 49-55
20. Bevan JA, Osher JV: A direct method for recording tension
changes in the wall of small blood vessels m vitro. Agents
Actions 1972;2:257-260
21. Lindvall 0, Borklund A: The glyoxylic acid fluorescence
histochemical method: A detailed account of the methodology for the visualization of central catecholamine neurons.
Histochemistry 1974;39:97-127
22. Cowen T, Haven AJ, Bumstock G: Pontamine sky blue: A
counterstain for background autofluorescence in fluorescence and immunofluorescence histochemistry. Histochemistry 1985;82:205-208
23. Marco E, Balfagon G, Candie MV: Indirect adrenergic effect
of histamine in human cerebral arteries: Influence of postmortem period. J Pharm Pharmacol 1984;26:253-255
24. Nystrom B, Olson L: Adrenergic innervation of human
cerebral arteries. Acta Neurochir (Wien) 1973;29:260-261
25. De la Lande IS, Waterson JG: Modification of autofluorescence in the formaldehyde-treated rabbit ear artery by
Evans-Blue. J Histochem Cytochem 1968;16:281-283
26. Saito A, Lee TJ-F: Serotonin as an alternative transmitter in
sympathetic nerves of large cerebral arteries of the rabbit.
Ore Res 1987;60:220-228
27. Shibata S, Cheng JB, Morakami W: Reactivity of isolated
human cerebral arteries to biogenic amines. Blood Vessels
1975;14:356-365
28. Hardebo JE, Hanko J, Kahrstrom J, Owman C: Electrical
field stimulation in cerebral and peripheral arteries: A critical
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
evaluation of the contractile response. J Auton Pharmacol
1986;6:85-96
Bevan JA, Bevan RD: Localized neurogenic vasoconstriction of the basilar artery. Stroke 1973;4:760-763
Lee TJF, Su C, Bevan JA: Neurogenic sympathetic vasoconstriction of rabbit basilar artery. Cine Res 1976^39:120-126
Laher I, Bevan JA: Alpha adrenoreceptor number limits
response of some rabbit arteries to norepinephrine. J Pharmacol Exp Ther 1985;233:290-297
Lee TJF: Ultrastructural distribution of vasodilator and
constrictor nerves in cat cerebral arteries. Ore Res 1981;
49:971-979
Araki A, Su C, Lee TJF: Effect of superior cervical ganglionectomy on the sensitivity of rabbit ear artery and cerebral
arteries of rabbit and cat to vasoactive agents. J Pharmacol
Exp Ther 1982^00:49-55
Duckies SP, Bevan JA: Pharmacological characterization of
adrenergic receptors of a rabbit cerebral artery in vitro. J
Pharmacol Exp Ther 1976;197:371-378
Bevan JA: A comparison of the contractile responses of the
rabbit basilar and pulmonary arteries to sympathetic agonists: Further evidence for variation in vascular adrenoreceptor characteristics..//>/tf!miflco/£<p Ther 1981;216:83-89
Hirst GDS, Neild TO: Evidence for two populations of
excitatory receptors for noradrenaline on arteriolar smooth
muscle. Nature 1980;283:767-768
Hirst GDS, Neild TO: Localization of specialized noradrenaline receptors at neuromuscular junctions on arterioles of
the guinea-pig. JPhysiol 1981;313:343-350
Neild TO, Zelcer E: Noradrenergic neuromuscular transmission with special reference to arterial smooth muscle. Prog
Neurobiol 1982;19:141-158
Bevan JA, Duckworth JW, Laher I, Oriowo MA, McPherson GA, Bevan RD: Sympathetic control of cerebral arteries: Specialization in receptor type, reserve, affinity and
distribution. FASEBJ 1987;1:193-198
Neild TO, Hirst GDS: 'The gamma connection': A reply.
Trends Pharmacol Set 1984;5:56-57
Bevan JA: "The gamma connection': Are we ready to throw
out t£e a-adrenoceptor in sympathetic vasoconstriction?
Trends Pharmacol Set 1984;5:53-55
Bevan JA, Su C: Variation of intra- and perisynaptic adrenergic transmitter concentrations with width of synaptic cleft
in vascular tissue. / Pharmacol Exp Ther 1974;190:30-38
Nosko M, Krueger CA, Weir BK, Cook DA: Effects of
nimodipine on in vitro contractility of cerebral arteries of
dog, monkey, and man. J Neurosurg 1986;65:376-381
Skarby T, Andersson KE: Contraction-mediating aadrenoceptors in isolated human omental, temporal and pial
arteries. J Auton Pharmacol 1984;4:219-229
Toda N, Okamura T, Shimizu I, Tatsuno Y: Postmortem
functional changes in coronary and cerebral arteries from
human and monkeys. Cardiovasc Res 1985;19:707—713
Brandt L, Ljunggren B, Andersson K-E, Hindfeld B: Individual variations in response of human cerebral arterioles to
vasoactive substances, human plasma, and CSF from patients
with ancurysmal SAH. /Neurosurg 1981;5:431-437
KEY WORDS • human • artery, middle cerebral • ianervation
• norepinephrine • a-adrenoceptor • extraceptor
Aminergic histofluorescence and contractile responses to transmural electrical field
stimulation and norepinephrine of human middle cerebral arteries obtained promptly after
death.
J W Duckworth, G C Wellman, C L Walters and J A Bevan
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Circ Res. 1989;65:316-324
doi: 10.1161/01.RES.65.2.316
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1989 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/65/2/316
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/