478
CIRCULATION RESEARCH
Tomanek RJ (1970) Effects of age and exercise on the extent of
the myocardial capillary bed. Anat Rec 167: 55-62
Turina M, Busaman WD, Rrayenbuhl HP (1969) Contractility
of the hypertrophied canine heart in chronic volume overload.
Cardiovasc Res 3: 486-495
VOL. 49, No. 2, AUGUST 1981
Unge G, Tomling G, Adollsson J, Ljungqvist A (1979) Capillary
proliferation in heart and skeletal muscles in swimming exercised and dipyndamole treated rats (abstr). Microvasc Res 17:
S22
Functional and Structural Changes in the
Rabbit Ear Artery after Sympathetic
Denervation
ROSEMARY D. BEVAN AND HIROMICHI TSURU
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SUMMARY We studied the tissue weight, dimensions, contractility, elasticity, and sensitivity to
exogenous norepinephrine (NE) of denervated «nd innervated segments of the central ear arteries of
white New Zealand rabbits. Three different age groups received unilateral superior cervical ganglionectomies, "growing" at 3-4 weeks, "young adult" at 9-11 weeks, and "mature" at 16-20 weeks. In the
growing group, 8 weeks after ganglionectomy, the denervated arteries showed mean decreases in
tissue weight (11%), total wall thickness (12%), cross-sectional area of media (17%), contractility (16%),
and increases in the tangential modulus of elasticity and sensitivity to NE (2.3-fold) compared to the
contralateral control vessels. The change in medial cross-sectional area was significant in the growing
and young adult but not the mature animals. The other changes, however, although consistently seen,
differed quantitatively among the groups. These results indicate that an intact innervation is necessary
for normal development and maintenance of the artery wall. However, the precise consequences of this
influence vary at different ages. Whether this influence involves a special trophic factor is not known.
CircRes 49: 478-485, 1981
DENERVATION produces remarkable morphological and functional changes in skeletal muscle
(Guth, 1968; Drachman, 1976). In several mammalian species studied, diversification of muscle fibers
occurs during postnatal development and is partially dependent on the innervation (Gutmann,
1976). It also has been reported that the sympathetic nervous system exerts a trophic influence on
skeletal and smooth muscle (Mendez et al., 1970;
Luco and Luco, 1971). However, it is generally
believed that'denervation of smooth muscle produces little morphological change (Thoenen and
Tranzer, 1963; Levi-Montalcini, 1972). More recently, several apparently conflicting observations
have been made regarding morphological changes
associated with denervation of smooth muscle, using different methods or species and tissues. Following sympathetic denervation of the growing rabbit
ear artery, there was less 3H-thymidine uptake
(DNA precursor) and there were fewer labeled
From the Department of Pharmacology. UCLA School of Medicine,
University of California. Los Angeles. California.
Suporledb\ US Public Health Service Grant HL 20581 and American
Heart Association-Greater Los Angeles Affiliate 4O8IG
Dr Tsuru's present address is: Department of Pharmacology, Nagoya
University School of Medicine, Tsuruma, Showa-ku, Nagoya 466, Japan.
Address for reprints Dr Rosemary D Bevan. Department of Pharmacology, UCLA School of Medicine, Los Angeles, California 90024
Received March 26. 1979, accepted for publication February' 10, 1981
smooth muscle cell nuclei in the tunica media than
in the control artery, suggesting that sympathetic
innervation influences proliferation of vascular
smooth muscle (Bevan, 1975). In contrast, Chamley
and Campbell (1976) observed that the presence of
sympathetic nerve fibers or homogenates of sympathetic chain delayed by several days the normal
dedifferentiation and proliferation of isolated medial smooth muscle cells from the aorta and ear
artery of "young" rabbits in tissue cultures. In addition, Campbell et al. (1977) have shown that
sympathetic denervation of chicken extensor secundariorum longus muscle by a total bracbial plexus
lesion in the 2-week and adult chicken produced a
significant increase in dry weight, due to hyperplasia of the smooth muscle cells.
In the present paper, effects of sympathetic denervation on contractility, dimensions, elasticity,
weight, and sensitivity to exogenous norepinephrine
were examined in the course of a study of sympathetic nervous influence on the blood vessel wall. A
preliminary report has appeared elsewhere (Bevan
and Tsuru, 1978).
Methods
Three groups of New Zealand white male rabbits
were used: (1) growing rabbits of 3—4 weeks of age,
(2) young adult rabbits of 9-11 weeks, and (3)
DENERVATION EFFECT ON VASCULAR SMOOTH MUSCLE/fleua/i and Tsuru
mature rabbits of 16-20 weeks. They were fed an
unlimited standard pellet diet.
Denervation of the Central Artery of the Ear
After sedation with chlorpromazine (10 mg/kg,
im), the rabbits were anesthetized by intraperitoneal injection of an anesthetic mixture containing
per 100 ml: chloral hydrate, 4.25 g; pentobarbital
sodium, 0.972 g; magnesium sulfate, 2.126 g; propylene glycol, 42.8% and alcohol, 11.5%. The dose used
was 2 ml/kg. The central artery of the ear was
denervated by removing the ipsilateral superior cervical ganglion under sterile conditions (de la Lande
and Rand, 1965).
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Tissue Preparation
Eight weeks after denervation, animals were
stunned by a blow on the head and rapidly exsanguinated. The mature rabbits were killed 10 weeks
following surgery. Both the denervated artery and
contralateral control ear artery were cut in situ by
use of three parallel blades held at a fixed standard
distance to obtain two segments of equivalent
lengths (3.0 mm) from each vessel. In addition,
segments were removed from both the proximal
and distal ends of artery adjacent to the measured
segments for the study of catecholamine histofluorescence and wall dimensions.
The preparations were submerged in Krebs bicarbonate solution [composition in mM: NaCl, 117;
KC1, 4.9; CaCl2, 1.6; MgSO4, 1.2; NaHCO3, 25; glucose, 11; calcium disodium ethylenediaminetetraacetic acid (EDTA), 0.025; and ascorbic acid, 0.11]
equilibrated with 95% O2 and 5% CO2, and dissection
was continued under a microscope to obtain clean
segments.
Catecholamine Histofluorescence
Whole mounts and transverse sections of the
adjacent proximal and distal segments from both
denervated and control ear arteries were used to
study catecholamine histofluorescence. Either the
fluorescence histochemical formaldehyde method
of Falck et al. (1962) or the glyoiylic acid method
of Lindvall and Bjorklund (1974) was used.
Tissue Weight
Artery segments were blotted on filter paper and
weighed on a Mettler balance (H54) as quickly as
possible prior to the in vitro studies. We compared
the tissue weights of 3-mm lengths, of the control
and the denervated arteries from identical sites, cut
in situ.
Internal Circumference and Wall Thickness
The internal circumference and total wall thickness were determined 1 hour after placing segments,
which had been cut open longitudinally, in Krebs
solution containing lidocaine (0.1 mg/ml) and
NaN0 2 (0.1 mg/ml). The flaccid pieces were blotted
on filter paper and laid flat, the adventitial surface
479
downward, on a glass slide. A microscope was used
to measure the internal circumference of the vessels
by use of a 10X micrometer eyepiece. The wall
thickness was determined by focusing the microscope (with a calibrated fine adjustment) at 100X,
first on the surface of the slide (on which the
adventitial surface of the vessel was placed) and
then on the internal surface of the vessel. The
difference, the vessel thickness, was read from the
fine focus. More than three measurements at different positions in the wall of each vessel were made.
Cross-Sectional Area of the Vascular Smooth
Muscle Layer
A 2-mm ring segment from identical sites of both
denervated and control ear arteries was fixed in 10%
buffered formaldehyde, dehydrated, and embedded
in paraffin. Five-micron transverse sections were
prepared and stained with hematoxylin and eosin.
With a calibrated micrometer, measurements were
taken of the diameter of the vessel to the innermost
and outermost layers of medial smooth muscle, in
two planes at right angles to each other. The approximate medial cross-sectional area was calculated.
Sensitivity to Exogenous Norepinephrine and
Contractility
Ring segments (3.0 mm in situ length) were used
for analysis of the sensitivity to exogenous norepinephrine and the contractility of the vessel.
Two stainless steel wires (o.d. 0.2 mm) were
inserted through the lumen; one was anchored to a
stationary support and the other connected to a
force displacement transducer (Statham G10B0.15-350). The preparation was submerged in a jacketed tissue bath (37 °C) containing 50 ml of Krebs
solution gassed with 95% O2 + 5% CO2. Tension
change was recorded on a Grass polygraph (model
79D). Resting tensions of 0.45 and 0.5 g were applied
to the denervated and the control arteries, respectively. These were derived from initial pilot active
tension length studies. After equilibration for 1
hour, the effectiveness of denervation was demonstrated by the absence of tetrodotoxin (TTX)-sensitive contractile response to transmural nerve stimulation (TNS) using biphasic pulses 0.2 msec in
duration and supramaximal voltage at 8 Hz from
Grass stimulator model S4.
After pretreatment with desmethylimipramine
(DMI, 3 x 10" 7 M), propranolol (3 X 10"7 M), and
hydrocortisone (8.7 X 10"6 M) for 30 minutes to
eliminate factors influencing ar-adrenoceptor sensitivity, neuronal and extraneuronal uptake, and /?adrenoceptor response (Furchgott, 1972), norepinephrine was added cumulatively to the bath until
the maximum response was obtained.
At the end of each experiment, the maximum
contraction was obtained using /-norepinephrine
(10~4 M), followed by histamine (NT3 M). Alternatively, Krebs solution was replaced with a depolar-
480
CIRCULATION RESEARCH
izing solution (composition in mM: K2SO<, 76; KC1,
10; KHCO,, 16; CaCl2, 2.5; MgCl 2 , 1.2; KH2PO<, 1.2;
and glucose, 5.6; Somlyo et al., 1969).
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Measurement of Elasticity
The elasticity of the vessel was determined from
the length-stress relationship. Length-stress curves
were obtained for 3.0-mm ring segments of ear
artery, set up as described above. Tissues were
equilibrated for 1 hour without load. The segments
were stretched in steps of 50 /zm every 5-10 minutes
by turning a fine screw adjustment attached to the
force displacement transducer. The distance between the outer edges of the wires suspending the
segment was determined at each equilibrium level,
through the tissue bath wall, using a microscope
with lOx micrometer eyepiece. This was regarded
as half internal circumference. Wall stress was expressed as passive stress (dynes/cross-sectional
area in cm2), for each experimental length.
Tangential moduli of elasticity (Bevan et al.,
1964) were calculated at the half internal circumference at which wall stress was equivalent to that
calculated to result from a blood pressure of 80 mm
Hg in the control artery (Birmingham, 1970).
Chemical Agents
The following drugs were used: /-norepinephrine
bitartrate (/-Arterenol Bitartrate, Sigma Chemical
Co.), desmethylimipramine HC1 (DMI: USV Pharmaceutical Co.), propranolol HC1 (Ayerst Laboratories), hydrocortisone sodium succinate (Solu-Cortef, Upjohn), lidocaine HC1 (Xylocaine, Astra Pharmaceutical Products, Inc.), histamine dihydrochloride (Calbiochem), tetrodotoxin (TTX; SankyoCalbiochem), and NaN0 2 (Mallinckrodt).
Statistical Analysis
Paired observations on the control and denervated ear arteries in each age group were compared
using the paired t-test. Differences between the
different age groups were analyzed by means of the
unpaired t-test (Dixon and Massey, 1957). An inference of statistical significance was made when P <
0.05.
Results
The mean body weights of growing, young adult,
and mature rabbits at the time of operation were:
(1) 410 ± 45 g (8) [mean ± SE, n = 8], (2) 2.30 ±
0.19 kg (8), and (3) 3.62 ± 0.40 kg (5), respectively.
At the time of final study mean body weights of
each group were: (1) 2.53 ± 0.14 kg (8), (2) 3.24 ±
0.33 kg (8), and (3) 3.81 ± 0.42 kg (5), respectively.
Denervation of one ear artery was confirmed in
all unilaterally sympathectomized rabbits by the
absence of catecholamine fluorescence at the adventitial-medial junction (Bevan, 1975). The inner vation pattern in the control ear artery was similar
VOL. 49, No. 2, AUGUST 1981
in brightness and distribution in all three age
groups.
Tissue Weight and Dimensions
When the data from individual rabbits were
paired for comparison in each age group, tissue
weight and total wall thickness were significantly
reduced in the denervated as compared with the
contralateral control vessel. The results are summarized in Table 1.
The only significant difference between the
weight and wall thickness of innervated vessel segments among age groups was in the wall thickness
of the young adult vessel, which was significantly
greater than that of the mature group (P < 0.05).
Weights of the denervated vessel segments expressed as a percent of control were in the growing
rabbit 89.05 ± 2.54% (8) [mean ± SE, n = 8] in the
young adult rabbit, 87.47 ± 3.03% (8), and in the
mature rabbit 84.08 ± 2.58% (5) (see Figs. 1 and 2).
The total wall thickness of the denervated vessels
expressed in the same way was in the growing rabbit
88.23 ± 2.27% (8), in the young-adult rabbit, 91.49
± 2.70% (8), and in the mature rabbit 93.37 ± 2.09%
(5). There was no significant difference in the percent decrease of tissue weight and total wall thickness of the denervated compared with the innervated vessels among the three groups.
Internal circumference of the vessel was measured in the young adult group. The values of the
control and the denervated vessels were 1.27 ± 0.08
mm (8), and 1.24 ± 0.08 mm (8), respectively. These
means were not significantly different.
Cross-Sectional Area of Media
Denervation was associated with changes in
cross-sectional area of the media. Expressed as a
percentage of control ear arteries, cross-sectional
area in the growing rabbits was 72.8 ± 5.48 (8), in
the young adult group 86.63 ± 4.14 (14), and in the
mature group 95.1 ± 5.38 (8), respectively (see Figs.
1 and 2). Thus, effects of denervation on the amount
of vascular smooth muscle in the vessel wall diminished with age. In the mature animal, there was no
significant decrease in cross-sectional area of the
media with denervation.
Responses to Transmural Electrical
Stimulation
The effectiveness of denervation, demonstrated
by the absence of catecholamine histofluorescence,
was confirmed by eliciting contractile responses to
electrical field stimulation. Stimulation frequencyresponse curves of vessels from the young adult
group are shown in Figure 3. Only at frequencies
greater than 2 Hz were consistent, but small, responses observed in the denervated vessel. The
contractile response of the denervated vessel at a
stimulation frequency of 16 Hz was 10.9 ± 3.5% (8)
DENERVATION EFFECT ON VASCULAR SMOOTH MUSCLE/Bewm and Tsuru
481
TABLE 1 Effects of Denervation on the Ear Artery Weight, Wall Thickness, Maximum Force Development,
Maximum Stress Development, Cross-sectional Area of Media, Norepinephrine ED-A and Tangential Modulus of
Elasticity in the Growing, Young Adult, and Mature Rabbits
Growing rabbita (n ™ 8)
Tissue weight (mg)
Total wall thickness (/im)
Young adult rabbits (n - 8)
Mature rabbits (n = 5)
Control
Denervated
Control
Denervated
Control
1.76±0.06
212±8.8
1.57±0.06f
188±11.2f
2.13±0.22
225±10.2
1.85±0.19f
207±9.4f
169±0.11
194±10.3
O.1276±0.0O75§ 0.1540±0.0077
Dener%rated
1.42±0.09f
180±5.7*
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Cross-sectional area of media
(mm 2 )
Maximum force development
0.1805±0.0150
2.25±0.32
1.90±0 34*
2.56±0.16
1.81±0.24t
2.42±0 13
2.07±0.11f
Maximum stress development
x 10" (dynes/cm 2 )
1.74±0.08
1.68±0.16
1.66±0.22
1.44±0.17f
2 05±0.05
1.88±0.07f
Norepinephrine E D B X
10" M
Tangential modulus of elasticity X 10* (dynes/cm 2 )
2.69±0.41
1.15±0.2O9§
6.57±1.74
2.81±1.59§
5.69±0.64
3.63±1.11*
5.32±0 23
7 99±1.22*
5.28±0.28
5.35±0.14
0.1310±0.0060§ 0.17O2±O.O114
O.1615±0 0132
Values represent mean ± SE. Growing, young adult, and mature rabbits were denervated at age 4 weeks, 9-11 Weeks and 16-20 week* respectively, and
studied 8 -weeks later, Statistical analysis was made by using the paired (test between control and denervated ear artery in each jroup. A statistical
inference of significance was made when P < 0 05
'P < 0.05, t P < 0.01. §P < 0.005, tP < 0.001. Values of tissue weight? are of 1 segment of 3 0 mm in situ length
that of the control (Fig. 3). This was not affected
by tetrodotoxin (TTX) 10"T g/ml, a treatment that
reduced the control response by a mean of 95.0 ±
3.1% (4) at 16 Hz. The patterns of the tetrodotoxinresistant responses of the denervated vessels in all
three age groups were similar.
Sensitivity to Exogenous Norepinephrine
Norepinephrine (NE) concentration-response
curves of innervated and denervated vessel rings
from the young adult group in the presence of
propranolol (3 x 10~7 M), desmethylimipramine (3
X 1CT7M), and hydrocortisone (8.7 X 10"* M) are
shown in Figure 4. The denervated vessel was 2.4fold more sensitive to NE than the control. The
denervated vessels of the growing and mature
gToups also showed an increased sensitivity to NE
of 2.3 and 1.6-fold, respectively (Table 1 and Fig. 1).
Maximum Contractile Response
The contractile response of the ring segments
induced by NE (10~4 M), followed by histamine
(10~3 M) or depolarizing K+ solution was considered
maximum. In a few instances, the contractile force
initiated by NE (HP4 M) was increased by 2 to 3%
by the addition of histamine (10~3 M). The response
to the depolarizing K+ solution was never more
than that to NE (HP4 M).
Although there was no significant difference in
the maximum force developed among the innervated vessels of the three groups, denervation resulted in decreased maximum force development at
all ages (Table 1). The maximum force development
of the denervated vessels expressed as a percentage
of control was: in the growing rabbit, 83.9 ± 5.34%
(8); in the young adult rabbit, 70.3 ± 7.3% (8); and
in the mature rabbit, 85.7 ± 1.84% (5).
Differences between the control and the denervated vessels became less marked (Table 1) when
corrected for cross-sectional area so that the maximum stress rather than the maximum force developed was compared. Although this parameter was
reduced significantly in the mature young adult
rabbits, this was not true of the growing group.
Length-Stress Relationship
As illustrated in Figure 5 for the young adult
group the length-stress curves of the denervated
vessels of all three groups were shifted to the left of
control. From each length-stress curve, tangential
moduli of elasticity were calculated at the circumference corresponding to a blood pressure of 80 mm
Hg in vivo calculated from the Laplace relationship.
Values from innervated vessels had a narrow range.
However, this parameter in the denervated vessel
was significantly increased in comparison to control,
indicating that the vessel wall was stiffer, i.e., less
distensible.
Discussion
These results indicate that long-term denervation produces structural and functional changes in
the central ear artery of growing, young adult and
mature rabbits. Some significant differences were
apparent when a comparison of the observed
changes in the various parameters among three
different age groups was made. Some of the changes
are found only after denervation in the growing
animal, whereas others are seen consistently at all
ages, although differing quantitatively among the
age groups.
Nonspecific postjunctional denervation supersensitivity of smooth muscle to agonists has been
recognized for many years (Trendelenburg, 1963).
CIRCULATION RESEARCH
482
VOL. 49, No. 2, AUGUST 1981
OENERVATED EAR ARTERY
M Denervated
Growing
lOOr
_l E
5 E -16
I
[ j | | Control
•21 3.0
.20
PERCENT OF CONTROL VALUES
j£
ult
20
i g i.o
O
j Mature
_j
-°<0.005
Ld
0
LU
D
0.025
P<.025 P<.005 P< 0O5
j 80--
rh
J
; 60-
300
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200
j
I
x<
-f-
100
0
/ > <o.ooi P<O,QQ\
iot-
20
- « 80
^t
*
30
S'oo
10
60
0
P< 0.005
^
^<0.005
P< 0.025
FIGURE 2 Comparison of measurements of paired
innervated and denervated ear arteries of growing rabbits
8 weeks after unilateral cervical ganglionectomy. All
values are mean ± standard error of the mean: (n = 8).
P refers to comparisons between denervated and innervated groups using paired t-test.
o
olOO
LJ
Z
5 50
1
g 0
/><0.05
FIGURE 1 Parameters of isolated denervated ring segments of ear arteries from rabbits of three age groups
are expressed as a percentage of their innervated controls. A unilateral superior cervicalgangtionectomy was
performed 8 weeks prior to testing. Et refers to the
tangential modulus of elasticity at internal circumference equivalent, to 80 mm Hg. For further details, see
text. Brackets refer to standard error of mean value (n
varies from 8-14). P refers to comparisons between denervated and innervated vessels using paired t-test.
In our studies, the denervated ear artery showed an
increased sensitivity to exogenous NE of 1.6- to 2.4fold, a value which is in agreement with previous
observations (Fleming et al., 1973) and of similar
order of magnitude to that found in the rat portal
vein following 6-hydroxy dopamine administration
(Aprigliano and Hermsmeyer, 1977). The NE ED50
of both innervated and denervated vessels increased
with age to maturity. In the present experiments,
drug sensitivity was determined in the presence of
propranolol, desmethylimipramine, and hydrocortisone to eliminate consequences of j6-adrenoceptor
activation, and neuronal and extraneuronal uptake,
which have been shown to influence the concentration of NE at a-adrenoceptors (Furchgott, 1972).
Other evidence of the increased sensitivity of the
denervated vessel to agonists is the increased magnitude of its contractile response to transmural
nerve stimulation in comparison to an innervated
vessel in the presence of tetrodotoxin (10~7 g/ml).
It has been well documented that TTX blocks
exclusively the nerve-induced contractile response
(Bevan and Su, 1975). Thus, the direct muscle
response was increased after denervation. In addition, the denervated ear artery was more sensitive
to K+-induced depolarization than the control (unpublished observation). This phenomenon is consistent with the conclusions of Fleming et al. (1973)
for the guinea pig vas deferens, and Aprigliano and
Hermsmeyer (1977) for the chemically denervated
ELECTRICAL STIMULATION
FREQUENCY-RESPONSE CURVE
l.5r
Control
Ear Artery
UJ
o
or
1.0
O
a
LU
CL
O
UJ
>
LU
Q
0.5
Denervoted
Ear Artery
C-L
4
8
16
32
STIMULATION FREQUENCY ( H z )
FIGURE 3 Steady state frequency-response relationship of the control and the denervated ear artery segments from young adult rabbits to transmural nerve
stimulation. Vertical bars refer to standard error of
mean: (n = 8).
DENERVATION EFFECT ON VASCULAR SMOOTH MUSCLE/Bevan and Tsuru
lOOr
Denervated
o
50
IO" 8
10"
icrT
io"6
I -NOREPINEPHRINE
icr 5
icr 4
(M)
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FIGURE 4 Cumulative norepinephrine concentrationresponse curves of the control and denervated ear artery
segments from young adult rabbits. Experiments were
done in the presence of propranolol (3 X 10'1 M), desmethylunipramine (3 X KF1 M), and hydro cortisone (8.7
x 7(T6 M). Vertical bars refer to standard error of mean:
(n - 8). '* P > 0.005. * P > 0.01.
rat portal vein, that the denervation-induced partial
depolarization of vascular smooth muscle cells contributes to their hypersensitivity (Fleming, 1976).
Among the three different age groups, with one
exception, there was no significant difference in
tissue weight and total wall thickness of the control
vessels—although there was a tendency for these
measurements to be greatest in the young adult
group. Denervated vessel segments in comparison
to their innervated controls showed mean decreases
in weight of 11-16%, and in wall thickness of 7-12%
(Table 1). As would be expected, in general, the
changes in the weight and total wall thickness were
comparable.
After chronic sympathectomy, the reduction in
cross-sectional area of the media is age related,
being greatest in the youngest group but not sig30
20
1 0
0 2
04
06
0.8
10
12
14
16
V 2 INTERNAL CIRCUMFERENCE (mm)
FIGURE 5 Passive length-stress curves of paired innervated and denervated ear artery segments from young
adult rabbits. Vertical bars refer to standard error of
mean: (n°=8).*P> 0.01. ** P > 0.05).
483
nificantly changed in the mature animal. Replication of vascular smooth muscle is marked in blood
vessels during the early postnatal period, decreases
with increasing age, and in the normal mature animal is infrequently seen (R.D. Bevan, unpublished
observations). Consequently, the greatest effect of
denervation on wall thickness would be expected in
the youngest group of animals provided the vascular
smooth muscle cell, once formed, is either retained
in the wall or else has a very slow turnover rate.
Our results are also compatible with previous observations that vascular smooth muscle proliferation was less in the 2-week denervated rabbit ear
artery at 6 weeks of age (Bevan, 1975). It is possible,
however, that the apparent differences in crosssectional area of the media, determined in vitro on
a histological section, reflect a change in wall structural components which results in an alteration in
the ability of the segment to retract after removal
from the body. However, the data do indicate that,
in all age groups, a structural change has occurred
as identical in situ lengths of vessels weighed less
after denervation and the change in total wall thickness was, in general, proportional to the change in
tissue weight. Although there was no significant
difference between innervated and denervated arteries in the internal diameter of the vessel in the
young adult group, this parameter was not measured in the growing group where the greatest
change in structure occurred.
Alterations in activity of the sympathetic nervous system have been shown to influence cell
proliferation in other tissues, the rat parotid gland
(Schneyer, 1974), and the epithelium of the small
intestine (Tutton, 1975). Electrical nerve stimulation increased cell size and number in the adult
parotid gland (Muir et al. 1975), and the mitotic
rate of the jejunal epithelial cells (Tutton, 1975). In
contrast, chemical and surgical sympathectomy resulted in prolongation of the jejunal cell cycle time
(Tutton, 1975) and a decrease in the number of
mitotic cells (Klein and Torres, 1978).
Chamley and Campbell (1976), observed that a
diffusible substance from sympathetic nerves delayed the onset of modification accompanied by
proliferation of cultures of smooth muscle cells isolated from "young" rabbit aorta and ear artery.
However, the processing of the tissues with trypsin
and collagenase to obtain isolated cells might well
have changed the membrane characteristics so that,
at this stage, they are not comparable to cells in
situ. These substances have been shown to stimulate cell division in fibroblast cultures (Blumberg
and Robbins, 1975). The sympathetic ganglia homogenate does appear to contain a factor enabling
a partial "repair" process to occur in the smooth
muscle cells, thus delaying the onset of dedifferentiation. The basis of differences in response to denervation of vascular smooth muscle in situ and in
culture, and also smooth muscle in other tissues,
remains to be elucidated.
484
CIRCULATION RESEARCH
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Maximum force development was decreased in
the denervated ear artery in comparison to its control (Table 1 and Fig. 1). When the force development had been converted to stress development by
correction for differences in cross-sectional area,
the differences between the control and the denervated vessels became less and, in the growing rabbit,
were not significant. In the growing rabbit, it seems
likely that the dimensional and contractility
changes result predominantly from the loss of vascular smooth muscle mass. However, the diminution in contractility cannot be accounted for by loss
of tissue mass in the young adult and in the mature
groups (Table 1 and Fig. 1). Thus, a qualitative
change in the contractile machinery may be involved in the loss of ability to develop tension after
denervation of these groups.
The functional and structural changes in the
artery wall after denervation could be attributed to
loss of a "trophic" factor or to the absence of
sympathetic drive. If the age-related effect of denervation on medial thickness could be extrapolated
to other blood vessels, the level of sympathetic
drive during growth might be a significant factor in
determination of blood pressure. This could have
both a genetic and environmental component.
In a genetic model of hypertension, the spontaneously hypertensive rat, blood pressure elevation
occurs mainly between 4 and 12 weeks of age, a
period of growth. There is evidence that transmission in a sympathetic ganglion from normal rodents
is not functionally mature before 3—4 weeks of age
(Black et al., 1971), although this may have regional
variations. In this animal model there is evidence
of increased peripheral sympathetic nerve activity
during growth (Judy et al., 1976; Nagatsu et al.,
1976) and, in the 6-week and adult animal, an
increased thickness of the medial smooth muscle
layer has been found in resistance vessels (Mulvany
and Halpern, 1977; Warshaw et aL, 1979). Although
many factors are known to induce proliferation in
vascular smooth muscle, it is interesting to speculate that it may be influenced by increased nerve
activity. Because vascular resistance is proportional
to the 4th power of the internal radius, even a small
change in medial thickness would have a pronounced effect on blood pressure.
The length-stress curves of the denervated vessels were shifted to the left in comparison to their
controls in all three groups. As seen in Table 1, it is
of interest that the values of the tangential modulus
of elasticity of the denervated vessels are inversely
proportional to age, although the control values are
similar. The shape of the length-stress curve of an
artery is complex, reflecting the relative importance
of the different structural components of the wall
to stretch at different tissue lengths. The observed
increase in elastic modulus probably is due to a
relative increase in extracellular material. The differences in the tangential moduli of elasticity of the
denervated vessels among the three different ages
VOL. 49, No. 2, AUGUST 1981
may reflect the relatively greater loss of vascular
muscle at younger compared to older age groups
combined with different changes in extracellular
components.
In conclusion, our data suggest that loss of sympathetic innervation may result in structural as well
as functional alterations in an artery. It is known
that the presynaptic innervation to the superior
cervical ganglion is not mature until 3-4 weeks of
age in the mouse (Black et al., 1971), which has a
temporal pattern of postnatal growth similar to that
of the rabbit. The ability of the sympathetic innervation to transmit impulses of high frequency is not
mature until 4-6 postnatal weeks in the rabbit
(Schweiler et al., 1970). It is likely that neuronal
activity would be more effective in influencing the
vascular wall only after the neuronal influence has
functionally matured. The sympathetic innervation
may either specifically influence cell replication or
may modulate metabolic processes in the vascular
smooth muscle cell. Age-related differences in the
various parameters investigated following denervation may reflect a change in the synthetic capacity and role of the vascular smooth muscle cell
during maturation of the arterial wall.
Acknowledgments
It is a pleasure to acknowledge the expert assistance of Irene
Jones.
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Circ Res. 1981;49:478-485
doi: 10.1161/01.RES.49.2.478
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