Am J Physiol Heart Circ Physiol 289: H608 –H613, 2005.
First published March 11, 2005; doi:10.1152/ajpheart.01290.2004.
Remote arteriolar dilations caused by methacholine:
a role for CGRP sensory nerves?
Naris Thengchaisri and Richard J. Rivers
Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland
Submitted 22 December 2004; accepted in final form 8 March 2005
calcitonin gene-related peptide; microcirculation; conducted vasodilation; muscarinic receptor agonist; intrinsic nerves
within the organ is controlled through
complex interactions that regulate arteriolar resistance and
define the paths of blood flow. The resistances of the entire
arteriolar network aggregate to define what is commonly
known as organ resistance. The specific resistance of an individual arteriole is controlled by a number of local variables,
including flow (17), pressure (19), metabolites (35), and neural
input (33), whereas the coordinated network response requires
vascular communication. Vascular communication is the process whereby areas of the vascular network adjust to the
vasomotor signals that are generated within the tissue or at
other remote locations along the vascular tree. It potentially
plays a role in the precise control of intraorgan blood distribution (30, 31). Although various efforts have been made, the
underlying mechanism of this vascular communication is not
fully understood (9).
BLOOD FLOW DISTRIBUTION
Address for reprint requests and other correspondence: R. J. Rivers, Dept. of
Anesthesiology and Critical Care Medicine, Johns Hopkins Univ., 600 N.
Wolfe St., Ross Rm. 351, Baltimore, MD 21287 (E-mail: [email protected]).
H608
Hyperpolarizing signals for vascular communication and for
remote control of arteriolar diameter reportedly pass from cell
to cell through gap junctions in the vessel wall (20). The length
constant for decay of this signal is much larger than can be
explained by the simple passive decay of an electrotonic signal
(6), and simple blockade of gap junctions does not abolish the
response (32). Therefore, additional pathways, such as nerves,
may be involved, or the signal may be regenerated within the
vessel wall as it moves from cell to cell. Because various
neural innervations of elusive function (12) have been identified on the arterioles in the cheek pouch and because previous
studies indicate that local anesthesia inhibits the vascular
relaxation response to acetylcholine (23), this study was performed to determine whether there may be a role for neural
mechanisms in the remote vasomotor signals generated by
arteriolar muscarinic receptor stimulation.
MATERIALS AND METHODS
Hamster Cheek Pouch
Vascular communication can be initiated by a number of methods
(4, 7, 24, 27, 30). In the present study, we initiated vascular communication by applying selective drugs onto an arteriole of the hamster
cheek pouch. In accordance with Institutional Animal Care and Use
Committee approved protocols, male golden hamsters (103–132 g
body wt) were anesthetized with pentobarbital sodium (70 mg/kg ip).
The cheek pouch was exteriorized for intravital microscopy (5) after
tracheotomy and insertion of an endotracheal tube. Continuous intraperitoneal injection of saline (0.008 ml/min) and anesthetic (0.075
mg/min) maintained the appropriate levels of hydration and depth of
anesthesia. The cheek pouch was continuously superfused with 37°C
physiological salt solution (PSS; in mM: 132 NaCl, 2.0 CalCl2, 1.2
MgSO4, and 20 NaHCO3) equilibrated with 0%, 5%, or 10% O2, 5%
CO2, and balance N2. Deep esophageal temperature of the hamster
was maintained at 37°C by a combination of conductive and radiant
heating.
The transilluminated pouch vasculature was viewed with a video
microscope (Micro Instruments model M2 microscope with Nikon
E400 components and an arm-mounted dissecting microscope, Witney, Oxon, UK). The microscope was coupled to a video (chargecoupled device) camera (model 72S, Dage-MTI, Michigan City, IN)
and a video monitor. Diameter was measured with a video caliper
(Texas A & M University Health Science Center) that was calibrated
with a stage micrometer, and diameter data were digitally recorded
(Work Bench).
Conducted Dilations Initiated by Muscarinic Receptor Stimulation
Methacholine (5 s, 100 ␮M) was applied by pneumatic ejection
from a micropipette (26). Micropipette application of a drug is a
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.
0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society
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Thengchaisri, Naris, and Richard J. Rivers. Remote arteriolar
dilations caused by methacholine: a role for CGRP sensory nerves?
Am J Physiol Heart Circ Physiol 289: H608 –H613, 2005. First
published March 11, 2005; doi:10.1152/ajpheart.01290.2004.—Remote vasodilation caused by arteriolar microapplication of acetylcholine cannot be completely attributed to passive cell-cell communication of a hyperpolarizing signal. The present study was undertaken to
ascertain whether a neural component may be involved in the remote
response. In the cheek pouch of anesthetized hamsters, methacholine
(100 ␮M) was applied to the arteriole by micropipette for 5 s, and the
arteriolar responses were measured at the site of application and at
remote locations: 500 and 1,000 ␮m upstream from the application
site. Superfusion with the local anesthetic bupivacaine attenuated a
local dilatory response and abolished the conducted dilation response
to methacholine. Localized micropipette application of bupivacaine
300 ␮m from the methacholine application site also attenuated the
remote dilation but did not inhibit the local dilation. Blockade of
neuromuscular transmission with botulinum neurotoxin A (1 U, 3
days), micropipette application of calcitonin gene-related peptide
(CGRP) receptor inhibitor CGRP-(8 –37) (10 ␮M) 300 ␮m upstream
from the methacholine application site, and denervation of the CGRP
sensory nerve by 2 days of capsaicin treatment reduced the conducted
dilation response to methacholine but did not affect the local dilatory
response. Together, these data support involvement of a TTX-insensitive nerve, specifically the CGRP containing nerve, in vascular
communication. Understanding the effect of regulation of a novel
neural network system on the vascular network may lead to a new
insight into regulation of blood flow and intraorgan blood distribution.
NEURAL CONTROL OF REMOTE ARTERIOLAR DILATIONS
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standard method we have used for many years. Specific application
controls were used to eliminate concerns about diffusion of the drug
to remote locations of interest (25). Arteriolar vasomotor responses
were sequentially measured at the site of drug application (local) and
500 and 1,000 ␮m upstream from the site of application (Fig. 1). Sites
were randomly viewed, and each response was measured at least
twice. The arteriole was always allowed to return to baseline before
the next measurement (ⱖ3-min delay between ejections).
receptor antagonist, was continuously applied by micropipette at the
arteriolar site 300 ␮m upstream from the methacholine application
site to test for a role of sensory nerves in the remote vascular
responses to methacholine. To verify the specificity of CGRP-(8 –37),
local arteriolar responses to adenosine, methacholine, and CGRP were
tested at the site of CGRP-(8 –37) application.
Neural Transmission Blockade
The role of CGRP sensory nerves was further investigated by
chemical denervation of the preparation with capsaicin (11). Capsaicin activates the vanilloid receptor on sensory nerves to cause an
increase in intracellular Ca2⫹ and a release of neuropeptide. The
receptor for capsaicin quickly desensitizes, but prolonged exposure to
capsaicin causes a neurotoxic effect and results in sensory nerve
denervation (16). Capsaicin (0.013 M in 10% alcohol, 100 ␮l) or
vehicle control was injected into the exteriorized cheek pouch of a
temporarily anesthetized hamster, and the animal was studied 2 days
later. Tissue samples were fixed after experimentation to verify
sensory nerve denervation. Arteriolar dilation responses to methacholine were compared between vehicle-control and capsaicin-treated
animals.
Calcitonin Gene-Related Peptide Receptor Blockade
Stimulated sensory nerves release calcitonin gene-related peptide
(CGRP), which causes vasodilation. CGRP-(8 –37) (10 ␮M), a CGRP
Immunohistochemistry
To confirm the effect of sensory nerve denervation, cheek pouches
from vehicle-control and capsaicin-treated animals were excised,
pinned onto dental wax, and then fixed in 2% formaldehyde and 0.2%
picric acid. Tissues were permeabilized by incubation in Triton X-100
(0.5%) in PBS (pH 7.4) for 30 min at 37°C and washed several times
in PBS. Whole mounts were incubated with blocking diluent (5% goat
serum and 1% BSA) at room temperature for 30 min and then with
rabbit anti-human CGRP polyclonal antibodies (1:200 dilution; Alpha
Diagnostic International, San Antonio, TX) at 4°C overnight. The
whole mounts were rinsed three times (5 min each) in PBS and then
incubated with secondary antibody (FITC-conjugated goat anti-rabbit
IgG, 1:200 dilution; Jackson Laboratory, Bar Harbor, ME) at room
temperature for 2 h. The whole mounts were rinsed three times (5 min
each) in PBS and then observed with fluorescent light microscopy
using our standard microscope and an intensified charge-coupled
device camera (model XR/MEGA10, Stanford Photonics). Images
were captured using a standard gain protocol, and 10 images were
averaged to minimize noise.
Chemicals
Bupivacaine, capsaicin, CGRP, CGRP-(8 –37), CsCl, methacholine, and sodium nitroprusside were purchased from Sigma (St. Louis,
MO) and BoTA from Allergan (Irvine, CA). The stock solutions (10
mM) of bupivacaine and capsaicin were first made in distilled water
and alcohol, respectively. The other chemicals were dissolved in PSS.
The subsequent concentrations of all chemicals were achieved by
dilution in PSS.
Data Processing and Statistical Analysis
For each treatment, at least two measurements were obtained from
a single vessel. The averages were used for further comparison.
Changes in diameter after the drug applications were compared at
each location using ANOVA with repeated measures or a compound
response model (JMP3.1.5, SAS Institute). P ⬍ 0.05 was considered
statistically significant. Values are means ⫾ SE.
Fig. 1. Schematic drawing of a cheek pouch arteriole and stimulation sites.
Methacholine is ejected from a micropipette to initiate a dilatory response.
Dilations are viewed at the application site and at 2 remote locations upstream.
Blockers of nerve transmission or neuropeptide receptor are applied between
local and remote locations by micropipette ejection or, alternatively, via the
superfusion solution. CGRP, calcitonin gene-related peptide.
AJP-Heart Circ Physiol • VOL
RESULTS
A total of 50 arterioles from 44 hamsters were used for data
collection. The average “maximal” diameter of all the methacholine application sites, determined by their responses to
methacholine (10 mM), was 43.2 ⫾ 1.5 ␮m, and the average
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Bupivacaine. Bupivacaine blocks neural transmission by blocking
Na⫹ channels, which are important for the transmission of action
potentials along the nerve. Bupivacaine is especially useful here,
because it can block Na⫹ channels that are not sensitive to TTX. TTX
is commonly used to demonstrate neural involvement, although sensory nerves possess Na⫹ channels that are not affected by TTX. To
test for the effects of local anesthetics, bupivacaine (100 ␮M) was
added to the superfusion solution for ⱖ20 min before the responses
were repeated. We found that bupivacaine caused some vasoconstriction and affected the local methacholine response. To eliminate these
confounding variables and to determine whether the effects of bupivacaine were specific to transmission of the signal to the remote sites,
we applied bupivacaine (10⫺7–10⫺3 M) by micropipette directly to
the arteriole 300 ␮m upstream from the methacholine ejection site.
Botulinum toxin. The role of a neural pathway was investigated by
inhibition of neural function with botulinum toxin. Botulinum toxin is
a zinc-dependent protease that selectively cleaves synaptosomalassociated protein-25, a component of the fusion complex mediating
synaptic vesicle exocytosis (1). This effectively blocks release of a
transmitter at the synapse. We infiltrated 1 U of botulinum neurotoxin
A (BoTox; BoTA) in 250 ␮l of saline into the exteriorized cheek
pouch of an anesthetized hamster and allowed the animal to awaken.
The vasomotor responses of arterioles to methacholine, sodium nitroprusside, and phenylephrine were tested in the usual fashion 3–7 days
after BoTA injection.
Zero-Na⫹ superfusate. Na⫹ is a critical ion for the transmission of
action potentials along the nerve. We verified the role of Na⫹
channels in the methacholine response by replacing NaCl with CsCl in
the superfusion solution. This brought the Na⫹ concentration in the
superfusion solution to 20 mM.
Sensory Nerve Denervation
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NEURAL CONTROL OF REMOTE ARTERIOLAR DILATIONS
baseline diameter, measured after equilibration with 5% O2 but
before any treatment, was 25.4 ⫾ 1.2 ␮m.
Neural Transmission Blockade
Fig. 3. Effect of localized inhibition with bupivacaine on the arteriolar dilation
response to methacholine. Bupivacaine (n ⫽ 4) was applied 300 ␮m from the
methacholine ejection site, and conducted vascular responses were observed
500 and 1,000 ␮m upstream from the methacholine ejection site.
Role of CGRP Sensory Nerves in the Arteriolar Dilation
Response to Methacholine
Because TTX does not block these conducted responses (3),
whereas bupivacaine does, an alternative explanation was
sought. Bupivacaine is reported to also block TTX-resistant
Na⫹ channels, which are found in sensory nerves (2). We
therefore tested for a role for sensory nerves in these remote
responses.
CGRP receptor blockade. Application of CGRP-(8 –37) 300
␮m from the methacholine application site significantly attenuated the conducted dilation response to methacholine but did
not affect the local dilatory response (Fig. 5A). To confirm the
specificity of CGRP-(8 –37), we tested the local dilatory response to adenosine, CGRP, and methacholine. CGRP-(8 –37)
inhibited the local dilatory response to CGRP but did not affect
the local dilatory response to adenosine and methacholine (Fig.
5B). CGRP is a potent vasodilator for hamster cheek pouch
arteriole (EC50 ⬃ 2 ⫻ 10⫺10 M; Fig. 5B, inset).
Sensory nerve denervation. CGRP sensory nerves were
chemically denervated by infiltration of capsaicin into the
cheek pouch, and the arteriolar dilation response to methacholine was examined 2 days later. The local arteriolar methacholine-induced dilation was not affected by capsaicin, but the
remote dilation response was significantly attenuated 500 and
1,000 ␮m upstream from the methacholine ejection site (Fig.
6A). Immunohistochemical images of hamster cheek pouch
confirmed that capsaicin decreased the presence of the CGRP
sensory nerve (Fig. 6B).
DISCUSSION
Fig. 2. Effect of superfused bupivacaine (100 ␮M) on arteriolar dilation
response to methacholine. Arteriolar responses to methacholine (100 ␮M)
were measured along the arteriole 0, 500, and 1,000 ␮m upstream from the
ejection site. *P ⬍ 0.05 vs. control.
AJP-Heart Circ Physiol • VOL
Our data provide evidence that vascular communication
initiated by methacholine stimulation involves activation of
CGRP sensory nerves. In contrast to the reported lack of effect
of TTX (8), our data show that remote dilations caused by
methacholine are inhibited by bupivacaine, BoTA, cesium,
capsaicin, and CGRP-(8 –37), suggesting involvement of the
CGRP sensory nerve in the remote dilation response. These
data strongly suggest that activation of methacholine receptors
leads to activation of a neural network and release of CGRP
onto the arteriole at locations remote from the methacholine
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Bupivacaine. The local anesthetic bupivacaine, applied to
the cheek pouch via the superfusate, led to a small decrease in
resting diameter: from 29.3 ⫾ 2.2 (control) to 23.6 ⫾ 1.4 ␮m
(with bupivacaine). Bupivacaine significantly partially inhibited the local dilatory response and completely abolished the
remote dilation response 500 and 1,000 ␮m upstream from the
methacholine application site (Fig. 2). The actions of bupivacaine suggested an important role for voltage-gated Na⫹ channels and involvement of the neural pathway in remote dilations
initiated by methacholine.
Bupivacaine affects local and remote responses when
applied via the superfusion solution. To dissect the effect of
bupivacaine on local vs. remote dilation responses, we
ejected bupivacaine onto the arteriole by micropipette 300
␮m upstream from the methacholine application site (Fig.
1). Interestingly, localized inhibition with bupivacaine significantly attenuated the conducted response 500 and 1,000
␮m from the methacholine ejection site without affecting
the local dilatory response to methacholine (Fig. 3).
Na⫹ depletion. To investigate the involvement of Na⫹
channels in conducting the signal along the nerve, we replaced
NaCl with CsCl in the superfusion solution. Interestingly, CsCl
significantly inhibited the local and conducted dilation responses to methacholine similar to superfusion with bupivacaine, further supporting involvement of Na⫹ channels in
remote dilation (Fig. 4A).
BoTA. To further confirm the involvement of a neural
response in the remote dilation response to methacholine,
BoTA (1 U, 3 days) was used to block neurotransmitter release.
BoTA did not alter the local dilatory response to methacholine
but significantly inhibited the remote dilation 500 and 1,000
␮m from the methacholine application site (Fig. 4B). The local
dilatory response to sodium nitroprusside (14.8 ⫾ 1.4 and
15.8 ⫾ 1.2 ␮m for vehicle control and BoTA, respectively, n ⫽
5) or local contraction response to phenylephrine (19.8 ⫾ 1.6
and 17.1 ⫾ 1.9 ␮m for vehicle control and BoTA, respectively,
n ⫽ 7) was not affected by BoTA.
NEURAL CONTROL OF REMOTE ARTERIOLAR DILATIONS
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Fig. 4. Effect of CsCl and botulinum toxin A
(BoTox) on the conducted dilation response
to methacholine. A: superfusion with CsCl
(n ⫽ 6) significantly inhibited local and conducted dilation. B: BoTox (1 U; n ⫽ 8),
infiltrated into the cheek pouch, did not affect
the local response but led to inhibition of
conducted dilation 500 and 1,000 ␮m upstream from the methacholine ejection site.
*P ⬍ 0.05 vs. control.
AJP-Heart Circ Physiol • VOL
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Fig. 5. Effect of CGRP-(8 –37) on the arteriolar dilation response to
methacholine. A: CGRP-(8 –37) was applied via micropipette to the
arteriole 300 ␮m from the methacholine ejection site. Remote dilation
responses to methacholine 500 and 1,000 ␮m from the CGRP-(8 –37)
ejection site were inhibited (n ⫽ 6). B: effect of CGRP-(8 –37) on the local
dilatory response to micropipette application of adenosine (10⫺3 M), CGRP
(10⫺6 M), and methacholine (10⫺4 M). CGRP-(8 –37) inhibited the local
dilatory response to CGRP (n ⫽ 6) but did not affect the local dilation
response to adenosine (n ⫽ 6) and methacholine (n ⫽ 6). Inset: CGRP
concentration (conc.)-response curve for arteriolar dilation when CGRP
was added to the superfusion solution. EC50 ⫽ 2 ⫻ 10⫺10 M (n ⫽ 6). *P ⬍
0.05 vs. control.
application site to enhance the remote dilation. The mechanism
whereby arteriolar muscarinic receptor activation generates a
neural signal is not known. There may be activation of vascular
muscarinic receptors with secondary transfer of a neurotransmitter from the vessel wall to the sensory nerve (22), or there
may be direct activation of muscarinic receptors on perivascular nerves themselves (21).
A role for neural networks in blood distribution has been
previously demonstrated in various vascular beds, including
intestine (10), brain (36), and heart (18). Release of vasoactive
substances during hypoxia (22) may play an important role in
initiating vascular communication during the hyperemic response. However, our data shed light on a potential neural
mechanism that is affected by muscarinic receptor activation.
This neural activation may coordinate blood flow distribution
by modulating the conducted dilation along the check pouch
arteriolar network. Consistent with our study, Figueroa et al.
(8) showed that electrical stimulation of the arteriole can cause
conducted vasodilation that is not affected by TTX. However,
TTX did prevent sympathetic nerve conduction caused by the
same electrical stimulation. In this case, it is possible that
TTX-insensitive sensory nerves were activated by the electrical
stimulation (2). Thus multiple mechanisms, including innervation of the sensory nerve, may play a role in remote vasodilatory responses.
Numerous studies suggest a mechanism for vascular communication that involves cell-cell communication of a hyperpolarizing signal. Nerve conduction involvement in remote
vasodilation complements the cell-cell gap junction mechanisms that have been previously described (8). In the present
studies, remote dilation in response to methacholine was only
partially attenuated by capsaicin denervation and the CGRP
receptor antagonist CGRP-(8 –37), leaving an ample residual
response that can be explained by other mechanisms, such as
cell-cell transmission of a hyperpolarizing signal.
It is most interesting to note that microapplication of CGRP(8 –37) and bupivacaine to the arteriole attenuated the dilation
responses farther upstream, beyond the direct site of drug
application. The drug did not need to be applied directly to the
area of the arteriole that was dilating to effect the response.
This suggests two possibilities: 1) Perivascular release of
CGRP causes conducted dilation beyond the area of neuropeptide release. This seems unlikely, however, because we were
unable to reliably demonstrate conducted dilation with direct
microapplication of CGRP. 2) CGRP receptor activation on the
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NEURAL CONTROL OF REMOTE ARTERIOLAR DILATIONS
arteriole may modulate cell-cell conduction rather than directly
dilate the vessel itself. So interruption of the neural mechanism
attenuates conduction of vasodilatory signals along the vessel
wall.
Acetylcholine and nitric oxide have been shown to enhance
conducted dilation (3, 6). The neural pathway we describe
offers one possible mechanism for this enhancement. Activation of the CGRP nerves by either substance would cause
release of perivascular CGRP, which would modulate cell-cell
conduction. This would cause less decay of the conducted
signal and potentially turn a decaying signal into a potentiated
signal. Variability in this CGRP mechanism could explain the
well-described variability in cell-cell conduction. In addition, a
recent study indicates that a similar neural mechanism plays a
role in the myogenic response (29). Thus perivascular nerves
appear to be playing a complex, critical role in the coordination
of blood distribution that is affected by perivascular humoral
agents as well as intrinsic properties of the blood vessel. This
means that direct activation of neural muscarinic receptors by
intrinsic acetylcholine [as suggested to be released near neuromuscular junctions (34)] may not be the most interesting
physiological process described by these data.
The effect of various muscarinic receptor antagonists on
methacholine-induced local and conducted dilation was previously reported from our laboratory (26). The M3 muscarinic
receptor antagonists had a higher equilibration constant (KB)
for the local response, and the M2 receptor antagonist had a
higher KB for the conducted response. Furthermore, the muscarinic M2 receptor has been shown in sensory neurons of rat
AJP-Heart Circ Physiol • VOL
dorsal root ganglion, I-B4-positive neurons, keratinocytes, and
endothelial cells (13). Thus it is possible that the muscarinic
M2 receptor is involved in the activation of the CGRP sensory
nerve to effect conducted dilation.
Clinical Implication
The CGRP-1 receptor has been identified in human coronary
artery (15) and bronchial blood vessels (14), indicating a role
for the CGRP nerve in coronary and bronchial vasculature. The
role of vascular conduction in coronary microvessels (28) has
been identified, but the role of CGRP in microvascular coordination requires further investigation.
In summary, this study has revealed a neural component to
the vascular communication initiated by muscarinic receptor
activation in the microcirculation of the hamster cheek pouch.
These data indicate that local coordination of the blood distribution may be actively modulated by an intrinsic neural network.
ACKNOWLEDGMENTS
Present address of N. Thengchaisri: Dept. of Companion Animal Clinical
Sciences, Kasetsart University, 50 Paholyothin Rd., Bangkok 10903, Thailand.
GRANTS
This study was supported by National Heart, Lung, and Blood Institute
Grant R01 HL-072922 and American Heart Association Grant-in-Aid
0455730U.
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Fig. 6. Chemical denervation of CGRP sensory nerve. A: conducted dilation response to
methacholine was significantly attenuated
after CGRP nerve denervation by infiltration
of capsaicin (10 ␮M, n ⫽ 6) into the cheek
pouch. Conducted dilation responses to
methacholine 500 and 1,000 ␮m upstream
from the methacholine ejection site were
inhibited. *P ⬍ 0.05 vs. vehicle control. B:
immunoreactivity against CGRP nerve was
decreased after capsaicin injection (n ⫽ 4).
There was some undefined binding, but there
are obvious decreases in perivascular neural
processes.
NEURAL CONTROL OF REMOTE ARTERIOLAR DILATIONS
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