794
Evidence for Hyperplasia in Mesenteric Resistance
Vessels of Spontaneously Hypertensive Rats Using a
Three-Dimensional Disector
MJ. Mulvany, U. Baandrup, and H.J.G. Gundersen
From the Biophysics Institute, Institute of Pathology, Stereological and Electron Microscopical Diabetes Research Laboratory, and Second
Clinic of Internal Medicine, Aarhus University, Aarhus, Denmark
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SUMMARY. Cellular dimensions in mesenteric resistance vessels from 10 spontaneously hypertensive rats and 10 Wistar-Kyoto rats have been determined using a random volume with an
unbiased counting rule as the counting unit (the disector). With this method, vessels first were
mounted on a myograph. Media thickness (spontaneously hypertensive rats, 11.3 nm; WistaiKyoto rats, 8.6 /*m; P < 0.01), lumen diameter (spontaneously hypertensive rats, 178 pm; WistarKyoto rats, 194 pm; P > 0.1), and maximum active wall tension response (spontaneously
hypertensive rats, 3.2 N/m; Wistai-Kyoto rats, 2.5 N/m; P < 0.05) were determined. After fixation,
serial sections normal to the long axis of the smooth muscle cells were made. In each vessel, the
disector was a defined volume of the vessel wall (volume ca. 25 X 103 FIM3) which was contained
in about eight of these sections. The number of nuclei within the disector was counted using an
unbiased, three-dimensional counting rule. On the basis that cells were mononudear (an assumption that was tested), the ratio of this number divided by disector volume equaled the numerical
cellular density. Measurement of the fraction of media taken up by smooth musde cells then gave
mean cell volume (spontaneously hypertensive rats, 563 /im ; Wistar-Kyoto rats, 615 /*m3; P >
0.1). From the myograph measurements, the number of cells per unit length (spontaneously
hypertensive rats, 10.4//im; Wistar-Kyoto rats, 7.4/^m; P < 0.05) and maximum force production
per cell (spontaneously hypertensive rats, 5.1 fiN; Wistar-Kyoto rats, 5.7 /iN; P > 0.1) could then
be calculated. The results suggest that hyperplasia, not hypertrophy, is the basis of the thicker
media of mesenteric resistance vessels from spontaneously hypertensive rats. (Circ Res 57: 794800, 1985)
IT is now well established that structural changes in
the vasculature are associated with hypertension
(Folkow, 1982). Essential hypertension is associated
with a decreased lumen diameter (Conway, 1963;
Takeshita and Mark, 1980) and an increased media
thickness (Suwa & Takahashi, 1971; Wiener & Giacomelli, 1983), and thus an increased wall-to-lumen
ratio. Similar results have been obtained in a variety
of hypertensive models, including the spontaneously hypertensive rat [SHR (Cox, 1979; Winquist
and Bohr, 1983)] and the two-kidney, one-clip Goldblatt renal hypertensive rat [RHR (Wolinsky, 1972;
Lundgren et al., 1974)]. Moreover, the increased wall
thickness is accompanied by an increased quantity
of smooth muscle (Mulvany et al., 1978; Mulvany
and Korsgaard, 1983). It is not however clear
whether this increased quantity of smooth musde is
due to hyperplasia (more cells) or to hypertrophy
(larger cells). In the aorta of the SHR, there is evidence for hypertrophy, associated with hyperploidy
(Owens and Schwartz, 1982). A similar hypertrophy
has been found in the aorta of RHR (Owens and
Schwartz, 1983). However, others have found that
the increased smooth musde quantity is assodated
with hyperplasia (Bucher et al., 1984). With regard
to smaller vessels, morphometric techniques have
indicated that there is hyperplasia in the SHR (Lee
et al., 1983; Mulvany, 1984a). Whether these differing results represent a true difference between different vessel types, or whether they are due to the
different techniques used, is not dear, because determination of partide number is notoriously difficult (Cruz-Orive, 1980). A new method for determining cell number in vascular smooth muscle
(Baandrup et al., 1985; Sterio, 1984) seemed, however, to offer a way of resolving the problem. Since
this method, using at least two parallel sections,
measures the number of cells in a random volume
[here termed the disector (Sterio, 1984)] of the material, it avoids many of the pitfalls assodated with
methods in which cell number is estimated from
independent random sections. In a limited investigation, we have therefore used this method to estimate cell number and mean cell volume in mesenteric resistance vessels of 20-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto
rats(WKY).
Methods
The characteristics of the rats used are shown in Table
1. From these, 2-mm-long segments of 3rd branch mesenteric resistance vessels were isolated. We examined one
vessel per rat, except for one SHR and two WKY, where
two vessels were investigated. The vessels were mounted
Mulvany et a/./Hyperplasia in SHR Resistance Vessels
795
TABLE 1
Characteristics of SHR and WKY
Age (wk)
Body wt(g)
Heart/body wt (mg/g)
Systolic blood pressure (mm Hg)
SHR
WKY
(n " 10)
(n = 10)
P
19.6 ± 0.2
351 ± 3
3.42 ± 0.05
171 ± 4
19.6 ±0.2
349 ± 3
2.87 ± 0.04
124 ± 2
<0.001
<0.001
NS
Results are expressed as mean ± SE. P values show significance level by two-tailed f-test. NS = ; not
significant; n = number.
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on a myograph as ring preparations, and were held in a
physiological saline solution at 37°C (Mulvany and Halpern, 1977). Under these conditions, there is no tone in
either SHR or WKY vessels (Mulvany et al., 1978). A
microscope was used to determine the media cross-sectional area, ah of the vessels (Mulvany et al., 1978). Vessels
then were set to internal circumference U, where L4 is 90%
of the internal circumference estimated for a transmural
pressure of 100 mm Hg [determined from the resting
tension-internal circumference characteristic (Mulvany &
Halpem, 1977)]. Normalized lumen diameter was taken
as lj = \JJT. The corresponding media thickness, m^ was
calculated assuming constant media cross-section (Mulvany, 1984b). The vessels were stimulated with control
activating solution (10 ^M noradrenaline in high potassium
solution), to determine the maximum contractile ability of
the preparation, denoted AT]. Thereafter, the bathing
solution was changed to calcium-free saline containing
5 DIM EGTA for 15 minutes, and then to a glutaraldehyde
fixative solution as previously described (Mulvany et al.,
1978). The vessels were then demounted, washed in buffered sucrose solution, postfixed in 1% osmium tetroxide,
processed by standard techniques, including block-staining with 2% uranyl acetate, and embedded in Epon. Using
the disector, the estimate of numerical density, and hence
of cell volume, is independent of orientation (see below).
However, for the estimation of mean cell and nuclear
lengths, the sectioning planes should be normal to the
long axis of the circumferentially oriented smooth muscle
cells. Therefore, before Epon was added, the vessels were
placed in a small quantity of agar (Janisch, 1974), and
vessel orientation was adjusted so that the subsequent
sectioning would be in planes parallel to the vessel axis
and perpendicular to the plane which had been contained
by the two mounting wires. The agar held the vessels in
this orientation when the Epon embedding medium was
applied. Vessels then were sectioned, and from a point
approximately half-way between where the mounting
wires had been, a series of ca. l-/im serial sections were
taken, placed on glass slides, and stained with toluidene
blue.
The method used to determine cell density has been
described in detail elsewhere (Baandrup et al., 1985). In
brief, for each vessel, a series of about eight consecutive
serial sections was used. These were photographed and
printed at a total magnification of about 1430. In the first
section (the 'top' section. Fig. la) an area, a^ of vessel
wall was delineated by two parallel lines approximately
perpendicular to the wall edges. A disector was defined
as the three-dimensional probe bounded by ad and the
top surface of the 'bottom" section. The disector has
volume vd = aid-hd, where hd = (s—l)-f, s = number of
serial sections, f = average section thickness. Within ad,
the number of nucleus profiles, n,, was determined, using
a modification of the 'forbidden line rule' [(Gundersen,
1977) i.e., nuclei transsected by the lefthand boundary are
not counted, all those transsected by the righthand boundary are counted)]. For this purpose, a nucleus was counted
"only if a portion of the translucent material within the
nucleus was visible in the profile (notice that the criterion
used for counting a nucleus does not affect the estimate
of numerical density). In the subsequent sections (e.g., Fig.
1, b and c), each of the n, nuclei was followed and marked)
and in the final section (the 'bottom' section), the number
still present was determined, rib (Rg- Id). The number rid
= (n,—rib) is then the number of 'downward-pointing*
nucleus ends within the disector when counting from top
to bottom. Please note that the number rid is independent
of the size of the nucleus, but depends solely on the
nucleus numerical density (Sterio, 1984). Therefore, on
the basis that each cell contains only one nucleus (see
below), an estimate of cell numerical density, N v , is given
by N v = rid/vd.
In the same manner, disectors in other parts of the
sections were defined and the cell numerical density in
these determined. About four disectors were analyzed per
vessel. Mean cell numerical density was defined as fl y =
Erw/ZVd/ where £ denotes sum from the four disectors.
The number of cells per unit vessel length then was
estimated from ai • Nv.
Further information was obtained by also determining
in the "top' sections: (1) Vv, the volume fraction of media
containing smooth muscle cells (determined by point
counting), and (2) the number of cell profiles, c, (using the
same forbidden line rule). From these results, the following parameters were determined: cell volume, vc = Vv/Nv;
nucleus length, U = (n^rid) • hd; mean cell active stress, oc
= ATi/(mj•Vv); cell length, U = (c^ru)•U; mean cell crosssectional area, ac = vjZ) mean force per cell, Fc = ova^
All results are presented as mean ± SE (number of
animals). Significance of differences between SHR and
WKY parameters was assessed by Student's two-tailed ttest. The variance of cell numerical density within vessels,
between vessels, and between SHR and WKY was tested
using a two-level nested analysis of variance (ANOVA)
(Sokal and Rohlf, 1969). Probability levels undeT 5% were
considered significant.
_
The average thickness of sections (t) was determined
as follows. In five blocks, 'steps' were cut, each step
consisting of 10 microtome passes. The blocks then were
placed on the stage of a light microscope, and the size of
the steps was determined with an ocular micrometer.
Average section thickness was calculated from step size
divided by number of passes and was found to be 1.05 ±
0.05 fim.
Results
The myograph measurements, the disector characteristics, the histologjcal determinations, and the
Circulation Research/Vo/. 57, No. 5, November 1985
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FIGURE 1. Serial longitudinal sections of rat mesenteric
resistance vessels, showing smooth muscle cells in
approximate cross-section. Nuclear profile 1 is an example of a nucleus contained in the top section (panel
a), but not in the bottom section (panel d). Profile 2 is
a cell where the nucleus is not present in panel a, but
is present in panel d. Profile 3 is nucleus present both
in panel a and panel d. The lines in panel a define the
counting area, a& of the disector. Using the notation
indicated in Methods, nuclear profiles of types I and 3
would be among those included in "n,," but only cells
of type 1 would be included in "n^'All cells within at
would be included in "c^" Note that, for this method,
the "nucleus" is defined as the central part of the
nucleus containing translucent material. The bar represents 50 urn.
derived parameters are shown in Table 2.
When mounted on the myograph, the media
thickness was increased by 31% and the media
cross-sectional area was increased by 26% compared
with the WKY vessels. The active force of the SHR
vessels in response to control activating solution was
26% greater than in the WKY vessels.
Using the disector, we found_no difference in the
mean cell numerical density, N v , of the SHR and
WKY vessels. As indicated in Table 3, the variance
of N v between animals was greater than expected
(P < 0.01), given the variance of the individual
estimates of cell numerical density, N v , within animals (coefficient of variation_= 35%). This indicates
that part of the variation in N v seen between vessels
was due to vessel heterogeneity, rather than to
inhomogeneity within vessels. On the other hand,
from the three animals in which two vessels were
analyzed, it appeared that the variance of cell numerical density between vessels within animals
(coefficient of variation = 40%) was not less than
that seen between animals (coefficient of variation
= 28%). Combining the estimates of Nv with the
myograph measurements, the number of cells per
unit vessel length was found to be significantly
increased (40%) in the SHR (Fig. 2a).
The measurement of media fraction occupied by
smooth muscle cells, Vv, was similar in SHR and
WKY vessels. The volume of smooth muscle cells in
the SHR and WKY vessels was therefore also similar
(Fig. 2b), as was their length, cross-sectional area,
and nuclear length. Combining the active force
measurements with the histological data indicated
that the active force per cell cross-sectional area and
the average force per cell were similar in the SHR
and WKY vessels.
Mulvany et a/./Hyperplasia in SHR Resistance Vessels
797
TABLE 2
Vessel Characteristics of SHR and WKY
SHR
(n = 10)
Myograph measurements
Media thickness, mi (/im)
Lumen diameter, lt (pm)
Media cross-section, ai (jim2)
Active tension, AT, (N/m)
11.3 ± 0 . 6
178 ± 8
6859 ± 577
3.2 ± 0.2
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Histological measurements
Volume fraction of SMC in media, Vv
Disectors:
Total volume, 2v d (jim' x 103)
Top sections: no. of cells, 2c,
no. of nuclei, Zn,
Bottom sections: no. of n, still present, Znt
Cell numerical density, 53V (/im"3 x 10"')
Average cell dimensions
Volume, vc (jim3)
Length, U (/jm)
Cross-section, a^ (/im2)
Average nuclear length, 1,, (/un)
0.821 ± 0.015
Combined myograph and histological measurements
No. of cells/segment length (per pm)
Active force: per cell cross-section, ac (kPa)
per cell, Fc (MN)
WKY
(n = 10)
8.6
194
5423
2.5
±
±
±
±
P
0.5
7
353
0.2
<0.01
NS
<0.05
<0.05
0.806 ± 0.017
NS
23.6
211
69
32
1.57
± 2.8
± 24
±9
±6
±0.14
29.5
185
54
28
1.38
± 5.2
± 26
±11
±8
±0.11
NS
563
41.1
14.1
13.4
±58
±2.9
± 1.4
±1.0
615
42.0
15.1
11.5
± 39
±4.1
± 1.4
± 1.1
NS
NS
NS
NS
10.4 ± 1.1
352 ± 20
5.0 ± 0.6
7.4 ± 0.6
376 ± 27
5.7 ± 0.9
<0.05
NS
NS
Results are expressed as mean ± SE. See text for methods of calculation. P values show significance
level by two-tailed /-test. NS = not significant, n = number. Blanks indicate that f-test is not relevant.
The method for determining cell numerical density requires that each cell contains one and only
one nucleus. This assumption was supported by our
observation that multinuclearity was not observed
in any of the ca. 4000 cells which were counted in
the top sections of disectors (£ct in Table 2) and
followed over 8 ^m. However, to test the assumption
more rigorously, three SHR vessels were fixed and
embedded in the normal way. They were then sectioned at an angle of about 60° to the long axis of
the smooth muscle cells. In this way, each cell was
contained within about five consecutive sections,
and could be followed throughout. We examined 33
cells, and in no case did we observe more than one
nucleus per cell.
Discussion
The method used here avoids one of the fundamental problems of stereology, that in a section it is
not possible to sample particles of different size with
equal probability: large particles will be seen more
frequently than small particles. Thus, even using
sophisticated methods to determine cell size, such
as computer reconstruction (Todd et al., 1983), the
method of cell selection favors larger cells, and a
correction must be made for the resulting error
(Cruz-Orive, 1980). OtheT methods of determining
cellular numerical density, such as estimating volume-to-surface ratios (Lee et al., 1983), or inferring
nuclear numerical density from the ratio between
frequency of nuclear observation and section thickness (Loud et al., 1978), requires assumptions about
the shape of the objects being measured. Similar
objections apply to methods which compare frequency of particles in one plane with their linear
dimension in a perpendicular plane (DeHoff and
Rhines, 1968; Aalkjaer and Mulvany, 1981). However, in the method we have used here, since we
are counting numbers within a randomly selected
volume, the numerical density determined must in
principle be an unbiased estimate. We may therefore
expect that our estimates of cell volume are smaller
than those previously reported. In this respect, it is
perhaps relevant that the cell volume measured (ca.
TABLE 3
ANOVA for CeU Numerical Density
Source of variation
SS
df
MS
F
P
Strain (SHR and WKY)
Within strains
Within animals
2.23
17.66
27.67
1
18
77
2.23
0.98
0.35
2.10
2.73
NS
O.01
Units are cells/(1000 /im5).
SS = sum of squares; df = degree f; MS = mean of squares.
798
Circulation Research/Vof. 57, No. 5, November 1985
a
20
SHR
WKY
i
i
CD
CD
-1:
10
1000
SHR
WKY
i
i
CD
e
|
500
O5
oo
o
FIGURE 2. Scattergrams showing (panel a)
number of cells per unit stgment length and
(panel b) mean cell volume in mesenteric
resistance vessels from 20-week-old SHR
and WKY. Bars show ± SE Vessels taken
from 10 SHR and 10 WKY.
CD
O
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0 *—
3
(
600 fim ) is in general somewhat smaller than that
previously estimated for rat vascular smooth muscle
cells [e.g., 2500 fim3, tail artery; 919 fim3, femoral
artery; 788 fim3, mesenteric artery; 399 jtm3, portal
vein (Todd et al., 1983); 700 /mi3, mesenteric resistance vessel (Mulvany, 1984a)].
The hypertrophy of aortic smooth muscle in renal
hypertension seen by Owens and Schwartz (1983)
has suggested that the response of smooth musde
to an increased load is, as for skeletal muscle (e.g.,
Hettinger, 1961) and cardiac muscle (e.g., Grossman
et al., 1975), to increase cellular content of contractile protein and, hence, cell size. Similar conclusions
may be drawn from the dramatic cellular hypertrophy seen in the portal vein when this is constricted
distally (Uvelius et al., 1981). In the SHR, the position is less clear, for although—as indicated in the
introduction—cellular hypertrophy has been reported in the aorta, in the smaller vessels there is
much evidence to suggest that there is hyperplasia.
The present investigation strongly supports these
reports. First, there was no indication that the SHR
cells were larger than the WKY cells: indeed on the
average they were 8% smaller. Second, from consideration of the variance of the determinations, there
was a less than 1% chance of the SHR cells being
large enough to account for our finding that the
media cross-section of the SHR vessels was 26%
greater than that of the WKY vessels. Third, on the
basis that all cells are mononuclear, we found that
the SHR vessels contained 40% more cells per unit
segment length than the WKY vessels. It may be
erroneous to assume that the cells are not multinucleated. However, two observations suggest that
they are not. First, no morphological study, to our
knowledge, has ever found multinuclearity in
smooth musde to be anything but a very rare event.
Second, our own investigation here, where all of 33
SHR cells were found to be mononuclear, indicates
that the chances of our estimate of a 40% increase
in cell number in the SHR vessels being due to this
percentage of nuclei being in multinudeate cells is
less than 1 X 10~7. Two other possible errors may
be considered. First, it is possible that the 'retraction
ratio* [i.e., the ratio of the vessel length when
mounted (which is not stressed in the longitudinal
direction) to the in vivo length] differs between SHR
and WKY vessels. Second, it is possible that fixation
affects SHR and WKY vessels differently. Although
neither of these possibilities can be definitively discarded, we have previously shown (Mulvany et al.,
1978) that it is unlikely that such errors have caused
any serious alteration in the comparative properties
of SHR and WKY vessels. With respect to the first
possibility, we found the unstressed lengths of segments from SHR and WKY to be the same, whereas
measurements of the in vivo length of segments
close to those we have used (Ichijima, 1969) were
also the same in SHR and WKY, suggesting a similarity in the retraction ratio of SHR and WKY vessels.
With regard to the second possibility, we found that
any shrinkage caused by fixation affects SHR and
WKY vessels equally. Taken together, therefore, the
results point strongly toward hyperplasia, not hypertrophy, being the cause of the increased media
thickness in the SHR vessels.
In drawing this conclusion, we emphasize that
the results are based on rather small sample sizes
from each vessel (as can be calculated from Table 2,
only about 0.2% of each vessel was contained within
the disectors). However, we find no reason to believe
(within the given 5% probability level of significance) that our condusion concerning hyperplasia is
erroneous. First, the selection of the position of the
disector in each vessel and the counting were performed without the investigator knowing the code
giving the strain of animal concerned. Therefore,
from a statistical point of view, the results can hardly
be in doubt. Second, although the disector volume
was small, the coeffident of variation of the resulting
estimates of the number of cells/segment length
between animals was only 30% (as can be calculated
Mulvany et a/./Hyperplasia in SHR Resistance Vessels
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from the data in Table 2). It is therefore unlikely
that this would be much improved even if the
disector volume were greatly increased. Thus, although it is always preferable to have as large a
sample size as possible, we believe that the size of
the samples used was sufficient to have provided a
useful result.
The stimulus for the hyperplasia is not clear, but
it does not appear to be increased pressure. In recent
experiments, we have found that the media thickness of mesenteric resistance vessels from SHR is
little affected if the animals are treated from age 4
weeks to 14 weeks with either hydralazine (Mulvany
et al., 1983) or felodipine (Nyborg and Mulvany,
1985) to keep them normotensive. On the other
hand, there is indirect evidence that the rate of
synthesis may be due to cellular factors which are
genetically determined (Kanbe et al., 1983). On this
basis, it is possible that the increased media thickness seen in SHR small arteries is due to a genetic
programming which ensures that the quantity of
smooth muscle cells produces a vascular structure
corresponding to the blood pressure which normally
develops in these animals. Thus, the vascular
smooth muscle cells in the SHR are not normally
overloaded, and therefore would not be expected to
receive a stimulus to increase cellular mass. In this
respect it would be interesting to determine whether
the increased vascular mass seen in mesenteric resistance vessels with renal hypertension (Mulvany
and Korsgaard, 1983) is the result of hypertrophy,
since this increase is more likely to be due to the
increased pressure.
In conclusion, our results indicate that the increased media thickness of mesenteric resistance
vessels in SHR is not associated with hypertrophy,
but, probably, with hyperplasia.
We thank Michall Stottze for excellent technical assistance.
This work was supported by the Danish Heart Foundation (Grant
7964) and the Danish Medical Research Council (Grant 12-4533).
Dr. Mulvany is affiliated with the Institute of Biophysics, Dr.
Baandrup with the Institute of Pathology, and Dr. Gundersen with
the Institute of Pathology, the Diabetes Research Laboratory, and the
Gink of Internal Medicine.
Address for reprints: Dr. M./. Mulvany, Institute of Biophysics,
University of Aarhus, DK-8000 Aarhus C Denmark.
Received October 23, 1984; accepted for publication August 20,
1985.
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INDEX TERMS: Hyperplasia • Hypertrophy • Resistance vessel
• SHR • WKY • Disector
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Evidence for hyperplasia in mesenteric resistance vessels of spontaneously hypertensive rats
using a three-dimensional disector.
M J Mulvany, U Baandrup and H J Gundersen
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Circ Res. 1985;57:794-800
doi: 10.1161/01.RES.57.5.794
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