Effect of Sympathetic Denervation on Smooth Muscle Cell
Proliferation in the Growing Rabbit Ear Artery
By Rosemary D. Bevan
ABSTRACT
The effect of sympathetic denervation on the uptake of 3H-thymidine (3H-Tdr) into the
ear artery of a growing rabbit was studied in vitro and in vivo. Uptake into the right artery
was compared with that into the left 2 and 3 weeks after left superior cervical
ganglionectomy in 4-week-old rabbits. Denervation was confirmed by the absence of
catecholamine fluorescence. The total uptake of 3H-Tdr was determined by scintillation
spectrometry, and its distribution in the artery wall was studied by light microscope
autoradiography. The denervated ear artery took up significantly less DNA precursor and
exhibited fewer labeled vascular smooth muscle cell nuclei in the tunica media than did
the control artery. These findings suggest that sympathetic innervation influences the
proliferation of vascular smooth muscle in growing rabbits.
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• During an investigation of the factors affecting
arterial smooth muscle cell proliferation, it became
desirable to determine whether the sympathetic
nervous system influences cell division and growth
of the artery wall in the developing animal. To
study the effects of sympathetic denervation on the
growing ear artery, a left superior cervical ganglionectomy was performed on 4-week-old rabbits. In
immature rabbits, appreciable numbers of arterial
smooth muscle cell nuclei are labeled after a single
injection (pulse label) of 3H-thymidine (3H-Tdr), a
DNA precursor, and mitotic vascular smooth muscle cells are frequently seen. The labeling index and
also the mitotic index of arteries studied slowly
declines after the first postnatal week. At 10 weeks
of age, the mean labeling index in the ear arteries
of 11 rabbits was 0.12% (unpublished results).
These results are in agreement with those of Spaet
and Lejnieks (1), who found a vascular smooth
muscle cell labeling index in mature rabbits of
0.16% after a 7-hour infusion of 3H-Tdr. The
normal mature rabbit appears to have a relatively
stable arterial cell population; labeled cells are
infrequent (2, 3), and mitotic vascular smooth
muscle cells are rare.
DNA synthesis and hence proliferative potential
in the growing rabbit was assessed by measuring
both the ability of normal and denervated ear
arteries to take up 3 H-Tdr in vitro 2 and 3 weeks
following sympathectomy and the labeling index of
vascular smooth muscle cells both in vivo and in
From the Department of Pathology, School of Medicine,
Center for Health Sciences, University of California at Los
Angeles, Los Angeles, California 90024.
This work was supported by Los Angeles County Heart
Association Grant 434-C1 and U. S. Public Health Service Grant
HD 05615 from the National Institutes of Health.
Received December 26, 1973. Accepted for publication April
27, 1975.
14
vitro. 3H-Tdr is utilized by cells in the DNA
synthesis (S) phase and incorporated rapidly into
DNA by a sequence of phosphorylation steps (4). It
is a more sensitive measure of proliferative activity
due to the relative lengths of these phases in the
cell cycle. In a brief labeling period, only cells in
the S phase will incorporate 3H-Tdr into nuclear
DNA, and the distribution of these cells can be
demonstrated by autoradiography. 3H-Tdr uptake
was measured by scintillation spectrometry.
Denervation resulted in significant reductions in
3
H-Tdr uptake into the artery and in the number of
smooth muscle cells synthesizing DNA. This finding suggests that the sympathetic nervous system
influences the rate of division of vascular smooth
muscle in the growing rabbit.
Methods
Male white New Zealand rabbits were fed an unlimited standard pellet diet. When they were 4 weeks old,
they were anesthetized with sodium pentobarbital (40
mg/kg body weight, ip), and their left superior cervical
ganglions were removed under sterile conditions. Sham
operations were performed on some rabbits; others were
not subjected to surgery.
At the fourteenth or the twenty-first postoperative day
at approximately 10 AM, the rabbits were killed by a blow
on the head and rapidly bled. Their ear arteries were
dissected out rapidly for an equivalent length and
submerged in cold, aerated physiological saline. Dissection was continued to obtain clean arteries under the
dissection microscope. Short portions of both the left and
the right ear arteries were freeze-dried and processed for
fluorescence microscopy using the Falck-Hillarp (5)
technique for catecholamines; 5/i sections of each artery
were examined under a Zeiss fluorescence microscope.
The remaining portions of normal, sham-operated, and
denervated arteries were then attached to plastic holders
and submerged in a jacketed tissue bath (37°C) containing 50 ml of physiological salt solution bubbled with 95%
O2-5% CO 2 .
Tissues were equilibrated for 30 minutes, and then
Circulation Research, Vol. 37, July 1975
15
NERVE EFFECT ON ARTERIAL MUSCLE GROWTH
3
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H-Tdr (specific activity 2-6 c/mmole, Schwarz Bioresearch) was added to the bath to make a concentration of
2 //c/ml. Cells in tissues removed from the body and
incubated in a physiological medium containing labeled
nucleosides at 37 °C appear to function for a short time in
a manner similar to that in vivo with respect to DNA
synthesis (6). After 20 or 40 minutes of incubation, the
tissues were rinsed with physiological salt solution at
37°C and washed for 20 minutes with two further
changes in bath fluid. The wash time for the removal of
loosely bound tritiated compounds had been determined
by a previous investigation.
Tissues were studied by autoradiography, scintillation
spectrometry, or both. For light microscope autoradiography, comparable portions of each artery were fixed in
Carnoy's solution, dehydrated in absolute ethanol and
dioxane, and embedded in paraffin; 5M sections were
placed on slides coated with liquid NTB2 Kodak emulsion in total darkness by the method of Kopriwa and
LeBlond (7). After drying they were placed with a
desiccant in light-proof Bakelite boxes and exposed at
4°C for 2 and 4 weeks. They were developed in Dektol
for 2 minutes at 17°C, fixed, washed, and stained with
hematoxylin and eosin. The total number of labeled
smooth muscle nuclei in eight alternate serial sections
after both 2 and 4 weeks of exposure was expressed as a
percent of the total number of smooth muscle cells observed.
For scintillation spectrometry, tissues were blotted
with filter paper, weighed, and digested; their radioactivity was then determined. A Nuclear-Chicago Mark II
scintillation counter was used. Uptake was expressed as
milliliters of bath solution cleared per gram wet weight of
tissue. This expression is equivalent to the tissue fluid
ratio.
Three groups of rabbits were studied. Group 1 consisted of 21 rabbits. Their vessels were assessed by
autoradiography and scintillation spectrometry after
exposure to 3H-Tdr (2 /ic/ml [specific activity 6.0 c/
mmole]) for 40 minutes. Group 2 contained 14 rabbits.
Their vessels were assessed by autoradiography after
exposure to 3H-Tdr {2 pc/ml [specific activity 2.0 c/
mmole]) for 20 and 40 minutes. To control the autoradiographic condition as accurately as possible, 5M sections
from control (right) and denervated (left) arteries were
placed in parallel on the same slide. Identical sets were
exposed to Kodak NTB2 nuclear emulsion for 14 and 30
days. Group 3 was made up of 3 rabbits. 3H-Tdr was
given by intravenous injection (2 jtc/g body weight
[specific activity 2.0 c/mmole]) 2 weeks after left superior cervical ganglionectomy; the rabbits were killed
1 hour later. The ear arteries were processed as described earlier for light microscope autoradiography.
Paired observations of 3H-Tdr uptake and the percent
of labeled smooth muscle cells in the right and left ear
arteries were compared in each individual rabbit using
the paired t-test of Dixon and Massey (8). A statistical
inference of significance was made when P < 0.05.
cence at the adventitial-medial junction (Fig. 1).
Fluorescence was invariably seen in sham-operated
left and right ear arteries. Occasionally, the denervated arteries were more difficult to dissect due
to increased periarterial fibrosis.
Results
Top: Catecholamine fluorescence in the adventitia of a control
right ear artery. Bottom: Corresponding denervated left ear
artery showing the absence of catecholamine fluorescence. Note
the nonspecific fluorescence of the internal elastic lamina and
the elastic fibers in the adventitia.
3
H-Tdr UPTAKE IN THE GROWING EAR ARTERY
Denervation resulted in a highly significant
reduction in 3H-Tdr uptake into the artery wall at
both 2 and 3 weeks after sympathectomy. The
results are summarized in Table 1. The mean
uptake after 2 weeks into the control (right) ear
artery of the sympathectomized rabbits was 1.71
ml/g wet weight of tissue. This value was not
significantly different from the uptake in the ear
arteries of the sham-operated and the unoperated
rabbits. The mean uptake into the denervated
arteries was 1.11 ml/g wet weight of tissue. This
reduction is highly significant (P < 0.001) when
the data are paired for comparison. A similar
change was noted at 3 weeks.
FIGURE 1
Denervation of the proximal portion of the left
ear artery was confirmed in all sympathectomized
rabbits by the absence of catecholamine fluoresCirculation Research, Vol. 37, July 1975
16
TABLE 1
Effect of Sympathetic Deneruation on 3H-Tdr Uptake into the Growing Rabbit Ear Artery
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17
NERVE EFFECT ON ARTERIAL MUSCLE GROWTH
TABLE 2
Effect of Sympathetic Denervation on the Percent of Smooth Muscle Cells Labeled with SH-Tdr in the Growing Rabbit Ear Artery
Smooth muscle cell labeling
Concentration
Mean paired
difference
(%)
VJL UUOC
of 3H-Tdr
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Group 1
Denervated
left ear
Control
right ear
Sham-operated
left ear
Control
right ear
Group 2
Denervated
left ear
Control
right ear
Group 3
Denervated
left ear
Control
right ear
20 minutes
40 minutes
20 minutes
P
40 minutes
P
-0.75(12)
<0.05
-0.22 (5)
0.6
-2.44(11)
<0.01
-0.64(3)
0.05-»0.02
2 jic/ml
1.40 ±0.45 (12)
1.73 ±0.32 (12)
2.94 ± 0.89 (5)
3.1 ±0.69(5)
2 Me/ml
3.15 ±0.63 (14)
3.92 ±0.66(11)
4.25 ±3.33 (14)
6.36 ± 0.85 (11)
-1.10(14)
<0.01
2 /ac/g body wt
0.65 ± 0.09 (3)
1.29 ± 0.07 (3)
All values are means ± SE; number of rabbits tested is given in parenthesis. Group 1 includes rabbits tested both 2 and 3 weeks after
ganglionectomy.
with 3H-Tdr (group 3) showed identical features,
although the cells were more lightly labeled and
fewer in number than those in group 2. This
difference was probably due to both a shorter time
of exposure of the blood vessels following a single
intravenous injection and an uptake of the isotope
into other tissues in group 3.
MITOTIC FIGURES
Mitotic figures appeared to be fewer in the
media of the denervated ear arteries than they were
in their controls. The total numbers, however, were
too small to allow a satisfactory statistical analysis
to be made.
Discussion
The results showed that the denervated rabbit
ear artery took up less 3H-Tdr and exhibited fewer
labeled nuclei in smooth muscle cells than did the
control artery. The number of smooth muscle cells
synthesizing DNA in the denervated artery was
50-74% of the control level in all groups. The
experiments were performed on 4-week-old rabbits
because at this time they are weaned and growing
rapidly. The pattern of sympathetic innervation at
this time appears to be similar to the adult pattern
when it is studied by the histochemical fluorescence method of Falck and Hillarp (5).
Circulation Research, Vol. 37, July 1975
The in vivo results confirmed the in vitro experiments. There is always great variation in the
absolute level of labeled cells in rabbits even from
the same litter at this age. However, each rabbit
served as its own control, and differences between
the denervated and the control ear arteries were
consistent and significant.
Following incubation the tissues were washed for
a period of time previously shown to remove most
loosely bound radioactive material. The seemingly
more significant difference in 3H-Tdr uptake in
comparison with nuclear labeling demonstrated by
autoradiography may be due to the presence of
unincorporated 3H-Tdr or phosphorylated forms in
the acid-soluble nuclear pool that was removed by
tissue fixative. However, since normal and denervated tissues were treated in an identical manner,
the data still indicate a greater uptake of 3H-Tdr
by normal tissue. There was no significant cytoplasmic labeling in either type of tissue over
background levels. Mitotic figures in smooth muscle cells could be seen in both denervated and
control arteries so some growth does occur in the
denervated artery. However, mitotic smooth muscle cells seemed fewer in the denervated artery, but
the numbers seen were too small to satisfactorily
quantify the difference. A small number of rabbits
showed no difference in the labeling indexes of the
18
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two ear arteries even though one artery was denervated. One rabbit had doubled its weight in 1 week
after a 2-week period of no weight gain. Both ear
arteries appeared more immature for their age and
showed high proliferation rates and many mitotic
figures. Other factors such as growth hormone or
nutritional status may have been involved in these
rabbits.
It is unlikely that changes in artery wall tension
were responsible for the experimental results. If the
blood pressure is constant, the Laplace formula
indicates that the greater the radius, the larger the
circumferential tension (9). The artery distends
after denervation so that lateral tension in the wall
should increase. Since increased tension is thought
to stimulate proliferation, any changes in wall
tension probably would have increased 3H-Tdr
labeling in the denervated vessels; the opposite was
the case.
In mice and rats, transection of preganglionic
cholinergic fibers of the superior cervical ganglion
impairs development of postganglionic neurons,
which exhibit reduced total levels of protein and
enzymes (10, 11). My results showed that the
sympathetic nervous system also appears to influence the effector organ during development, in this
case, arterial smooth muscle cell proliferation. I
was not able to determine whether the rate of
uptake of 3H-Tdr was reduced after denervation,
since the nuclei were heavily labeled at both in
vitro incubation times and the number of DNA S
phase cells was relatively small. Neither could I
determine whether denervation delayed growth
and maturation of the artery or whether it had a
permanent effect on the size of the artery and the
metabolism of the vascular smooth muscle cells.
Additional studies must be undertaken to investigate these points.
Trophic effects of innervation on development,
differentiation, and maintenance of peripheral
structure have been studied extensively in voluntary muscle by many investigators, and reviews by
Gutman (12) and Guth (13) have been published.
Nerves are essential for the development and
differentiation of voluntary muscles, and their
absence during morphogenesis leads to atrophy.
Nerves affect or regulate muscle cell metabolism;
they play a role in protein and enzyme synthesis
and degradation and in glycogen metabolism. In
their absence, pronounced morphological, physiological, and biochemical changes occur in striated
muscle. Experiments by other investigators point
to a trophic role for the sympathetic nervous system on postsynaptic systems. Luco and Luco (14)
BEVAN
have found a depression of fibrillation in denervated striated muscle after reinnervation with
sympathetic postganglionic adrenergic fibers that
is not mediated by norepinephrine or acetylcholine.
Recently, Ramirez and Luco (15) have shown that
preganglionic sympathetic axons can reinnervate
voluntary muscle and have a trophic effect resembling the motor nerve supply to slow muscle.
Dresden (16) and Singer and Caston (17) have
reported that synthesis of protein, DNA, and RNA
declines after denervation in the early stages of
regeneration of a limb of the newt. The latter have
shown that incorporation of the precursors 3H-uridine and 3H-Tdr, after an initial short rise, falls to
a plateau of 50-60% of that on the normal side. My
results indicated that incorporation of 3H-Tdr fell
to 50-74% of normal in the denervated ear artery.
Evidence for a /3-receptor-activated mechanism
initiating DNA synthesis in hemopoietic stem cells
has been presented by Byron (18). On exposure in
vitro to very low concentrations of the /3-adrenergic
stimulant, isoproterenol, the resting cells commence synthesizing DNA. This effect can be
blocked by the specific /8-receptor blocking agent,
propranolol. Byron (18) has also demonstrated that
cyclic AMP stimulates DNA synthesis. Since 0adrenergic activity is associated with activation of
the adenyl cyclase system (19), which is found
predominantly in the cell membrane, a possible
mechanism of activation of DNA synthesis does
exist. It is of interest that /3-receptors have been
shown to be present in almost all vascular smooth
muscle cells. However, sympathetic stimulation
may affect ATP concentration. ATP is essential as
a phosphate donor in the phosphorylation of thymidine and has been shown to be an activator of the
enzyme involved in the synthesis of deoxyribonucleoside triphosphate, particularly at the level of
the ribonucleoside diphosphate reductases (20).
This enzyme has a differential time course of
activity in different tissues during development
and shows increased activity during periods of
intense cell proliferation. DNA replication in microbial systems has been shown to be stimulated by
ATP (21, 22).
Isoproterenol has also been shown to have a
differential age-dependent effect on the rat submandibular gland (23). During the first week of
postnatal life, isoproterenol causes hypertrophy
and hyperplasia by stimulating acinar cell precursors. Regression occurs after cessation of treatment. In rats over 2 weeks old, isoproterenol
stimulates DNA synthesis, mitotic activity, and
protein synthesis of acinar cells, causing hypertroCirculation Research, Vol. 37, July 1975
19
NERVE EFFECT ON ARTERIAL MUSCLE GROWTH
phy and hyperplastic enlargement of the gland
(24). Cavallero et al. (25) have found that in the
mature rabbit norepinephrine stimulates DNA
synthesis in the aorta and the pulmonary arteries.
Norepinephrine acts on both a- and /3-receptors.
Although it is probable that many factors affect
proliferation of vascular smooth muscle cells in the
growing animal, these experiments suggest that the
sympathetic innervation exerts a significant influence on this process in muscular arteries. It is
possible that the effect is mediated through the aor /3-receptors of the smooth muscle cells.
11.
12.
13.
14.
15.
Acknowledgment
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The author gratefully acknowledges the technical assistance
of Ms. Karen Luebbers and Ms. Mary Su and the secretarial
assistance of Ms. Elizabeth Ainley.
16.
17.
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Effect of sympathetic denervation on smooth muscle cell proliferation in the growing rabbit
ear artery.
R D Bevan
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Circ Res. 1975;37:14-19
doi: 10.1161/01.RES.37.1.14
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