681
Fabric Organization of the Subendothelium
of the Human Brain Artery by
Polarized-Light Microscopy
H.M. Finlay, J.G. Dixon, and P.B. Canham
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The thickened subendothelium of brain arteries that is characteristic of atherosclerosis was
assessed for the directional organization of the two main birefringent components, smooth
muscle cells and collagen. Thirty-three arteries from 16 autopsy cases were pressure fixed at 30,
60,110, and 200 mm Hg, sectioned at a thickness of 7 pm, and stained with silver impregnation
to enhance tissue birefringence. The intended focus of the study was on muscle organization,
but it also included the collagen among the cells because of the coalignment of the two tissues
and their similar staining properties for polarized-light microscopy. The birefringent medial
fabric at all pressures was circumferentially oriented, with a mean deviation of the 33 sections
of 1.4° from circumferential with an average circular standard deviation of 3.5°, thereby
showing remarkable coherence. In contrast, the subendothelium showed great variability both
in thickness and in organization. Many arteries had no measurable subendothelium, and
others had as much as 100%, with some atherosclerotic lesions as much as 300% of the medial
width. Measurements from the subendothelium revealed a helical arrangement of tissue, often
divided into separate regions, with a balance of left- andright-handedhelical components and
generally with lower pitch angles in the layers adjacent to the lumen. The average circular
standard deviation within individual subendothelial layers was 14.5°. (Arteriosclerosis and
Thrombosis 1991;ll:681-690)
D
iffuse intimal thickening is a characteristic
component of atherosclerosis in many major arteries and can have devastating consequences as a contributor to cerebral vascular disease and stroke. The proliferation of smooth muscle
tissue is an integral part of this process, and the
birefringent optical properties of smooth muscle
have made possible the assessment of its directional
organization in the subendothelium. Measurements
obtained by this method have revealed more order in
the intima than may have been previously appreciated,
which leaves open the possibility that this part of the
vessel wall may contribute mechanical function.
A necessary condition for smooth muscle tissue to
fulfill its mechanical function is that its cells be
organized with a common direction of alignment.
That this condition is met for the medial layer of the
From the Department of Medical Biophysics, University of
Western Ontario, and The John P. Robarts Research Institute,
London, Canada.
The authors acknowledge, with thanks, the Heart and Stroke
Foundation of Ontario for the research support through a term
grant to P.B.C.
Address for correspondence: H.M. Finlay, Department of Medical Biophysics, University of Western Ontario, London, Ontario,
Canada N6A 5C1.
Received June 11, 1990; revision accepted January 29, 1991.
arteries of heart and brain is well supported from
descriptions made several decades ago.1-3 Quantitative alignment studies by polarized-light microscopy
have substantiated those early findings.4-5 A similar
morphometric study applied to the subendothelium
might establish if that layer, which with time becomes
a characteristic component of the larger brain arteries, also has the potential to be a contributor to the
mechanical behavior of those arteries. Research that
concerns mechanical function of arteries has focused
on the tunica media, the layer considered to be the
most significant mechanically,6 and there the role of
smooth muscle cells is well documented. The cell is
the primary source of connective tissue components
in the media78 and also serves the mechanical function of providing muscle tone against the forces of
blood pressure.9-10 However, in the region of the
subendothelium, which is a layer of negligible dimensions in young arteries but whose dimensions increase with age,11 injury,12-13 and diet,14 the major
research thrust has been toward the cell's involvement in producing vessel wall components associated
with atherosclerosis.15"17
The intended focus of this study was on muscle
organization; however, the similar staining quality of
both muscle and adjacent collagen, their similar
682
Arteriosclerosis and Thrombosis Vol 11, No 3 May/June 1991
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alignment, and the variable and often low concentration of muscle cells in the subendothelium indicate
that collagen organization has been included in the
measurements. In our investigation, we have taken
advantage of the birefringent optical properties of
smooth muscle18 and of reticular collagen19 to measure structural organization by use of the polarizedlight microscope. In previous work, we established
the reliability of a method using the four-axis universal stage for vascular muscle cells4 and medial collagen.20 With this attachment to the polarizing microscope, we were able to make three-dimensional
measurements of orientation with a precision of
±2.4° for muscle cells and of ±2.9° for collagen of the
tunica media of brain arteries. The reference for
alignment is the edge and surface of the microscope
slide, which makes possible later transformations, so
that data can be reported in relation to the central
axis of the original artery. The technique has been
applied to two layers of the cerebral artery, the
collagenous tunica adventitia,21 and the smooth
muscle4 and collagen20 of the tunica media, each of
which is aligned mainly circumferentially. The subendothelial layer is relatively thin and has a more
varied structural organization. Studies of the rat
aorta, which is an elastic artery, describe the subendothelium as predominantly longitudinal for the
deeper region of the subendothelium and more
helical for the luminal side.22 We endeavored to
seek out a regular pattern to the organization of the
subendothelium and to focus on the group of
arteries associated with the circle of Willis that are
prone to vasospasm. We undertook, too, to assess
the variability of the subendothelial layer that might
arise from changes in transmural pressure. For this
purpose, arteries were fixed at static transmural
pressures of various magnitudes from 30 to 200 mm
Hg. Despite the increasing circumference with increasing pressure, we did not reveal any unifying
trend with pressure regarding the highly varied
subendothelium. The average orientation of the
tunica media remained remarkably circumferential
over the wide range in pressure.
Methods
Tissue Preparation
Thirty-three arteries were obtained after autopsy,
either from whole brains that had been fixed by
pressure perfusion or as segments of vessels that
were ligated, cannulated, and fixed under static
transmural pressure of 4.0, 7.9, 14.5, or 26.4 kPa (30,
60, 110, or 200 mm Hg). Tissue was taken from 16
persons aged 42-85 years, nine men and seven
women. The distribution of arteries used for the
medial study was seven vertebral, seven basilar, 14
middle cerebral, three anterior cerebral, one superior cerebellar, and one posterior cerebral. For the
subendothelial study, the same arteries were used
except for seven middle cerebral, one superior cerebellar, and two anterior cerebral arteries, all of which
had insufficient subendothelial thickness. The fixative
used was either 10% neutral-buffered formalin or
Karnofsky's perfusion fluid.23 Segments were dehydrated through increasing concentrations of alcohol,
were processed for paraffin embedding, and were
sectioned at a 7-/xm thickness. Long, straight portions of vessels were selected to ensure as much as
possible that the vessels were cut in cross section. For
some vessels, a miter box arrangement was used24 in
which the wax-embedded artery segment was positioned, cut, and flat mounted for sectioning. The
sections were stained with Gomori's silver impregnation25 or James' silver,26 which enhances the natural
birefringence of smooth muscle, causing it to appear
a bright yellow when viewed microscopically with
polarized light, as opposed to the adventitial collagen, which appears pink-orange. Silver is also a
reticulin stain, reticulin being that finer fabric of
collagen associated with smooth muscle cells (e.g.,
dog intestinal smooth muscle27). We have explored
rat bladder tissue with sequential slides of tissue
stained with one of the following: 1) Gomori's one-step
trichrome28 (for bright-field microscopy, showing
smooth muscle cells as dark red with light green-blue
extracellular connective tissue); 2) silver impregnation;
or 3) Picrosirius Red, a birefringent-enhancement stain
specific for collagen with negligible enhancement for
smooth muscle cells.29 The rat bladder, with submaximal distension, reveals regions of closely packed
smooth muscle cells (similar to arterial tunica media)
with coaligned straight, fine collagen (the reticulin
fibers) and large surrounding fields of unstraightened
collagen (similar to arterial adventitia). By this method
of comparing and cross checking, we have clarified that
the silver staining strengthens the birefringence of the
fibers of both reticulin and smooth muscle. In regions
dominated by smooth muscle (as in the tunica media),
the measurements would have been primarily due to
muscle cell alignment. For the present study, the subendothelial measurements were made from regions of
widely varying fractions of smooth muscle. Adjacent or
nearby sections to those used for measurements were
stained with Gomori's one-step trichrome and were
used to assess the relative concentrations of smooth
musclefibers.The subendothelial layer of brain arteries
was found to contain a higher proportion of smooth
muscle than that in our assessment of coronary arteries,5 with the exception of the areas of localized intimal
thickening where the smooth muscle was less concentrated, and in some areas, very sparse.
Method of Measurement
The ordinary polarizing-light microscope has a rotating stage that permits the recording of azimuth, or
rotation angle, relative to an orientation reference on
the tissue section. The additional requirement for a
unique orientation in three dimensions is an angle of
elevation relative to the plane of the section. The basic
procedure for making three-dimensional orientation
measurements has been adapted from geological science30 for smooth muscle tissue.4 We used a Zeiss
Finlay et al
Inner
Stage Axis
Brain Artery Subendothelial Orientation
683
Microscope
, Axis •
\
FIGURE 1. Schematic of universal stage, showing four
rotational axes. (Redrawn from Phillips30; reprinted
from Finlay et al,2* with permission of the Journal of
Microscopy.,)
North-South
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Universal polarizing microscope with a four-axis universal stage, which allows rotation of the specimen slide
out of the plane of the microscope stage. The principal
features of the stage, shown in Figure 1, are the degrees
of rotational freedom, the measuring arcs for reading
the elevation angle, the glass hemispheres, and the
coating medium of glycerol, the latter two which, in
combination, eliminate reflections from oblique airglass interfaces, and long-working-distance, low-numerical-aperture objectives (not shown). A high degree
of precision is achieved because measurements are
obtained at the position of extinction for the small zone
of tissue being assessed. Extinction occurs when the
optic axis of the birefringent fibers is aligned in the
plane of polarization of the polarizing filters. Any
deviation from this precise alignment results in the
transmission of light and loss of extinction. (We have
demonstrated the quantitative dependence of light
transmission between cross polars for highly oriented
collagen fibers.24)
Measurements were taken from both the medial
and subendothelial layers at regularly spaced intervals around the circumference on each artery section.
We found that in several vessels there was insufficient subendothelium to make measurements, and in
others the layer was incomplete. The larger arteries
such as the basilar and middle cerebral arteries were
more likely to have a subendothelium that could be
assessed. Polarized-light photomicrographs of two
different artery sections stained with James' silver are
shown in Figure 2. At this orientation, the medial
layer reveals strong birefringence. The demarcation
of the layering in the subendothelium of Figure 2, left
panel, is particularly emphasized, with the darker
region being indicative of a fabric lying at a highelevation angle to the tissue section. When making
measurements of the subendothelial layer, we
noted that readings immediately adjacent to the
internal elastic lamina often contained a high radial
component of orientation, which we judged to be an
East-West
interference artifact from elastin. Therefore, we
avoided taking readings immediately adjacent to
the internal elastic lamina, so that although individual measurements are from a zone of approximately
4 pm, our practicable limitation for the thinnest
layer of subendothelium measured radially was
10-15 /im. Since endothelial cells do not show any
significant birefringence, there was no such interference at the inner boundary.
Graphic Presentation of Data and Analysis
The measurements of orientation were plotted on
a Lambert equal-area projection, a mapping technique that permits three-dimensional orientation
vectors to be plotted on a graph.31 Examples of
Lambert projections showing medial and subendothelial layers from two arteries are shown in Figure 3,
along with tracings of the vessels from which the
measurements were made. On these projections, the
circumferential reference is located at the center,
giving the least distortion to the mainly circumferential orientation of the medial layer. Helically oriented
smooth muscle would be displaced on the vertical
axis, with longitudinal fibers shown at the northsouth poles. A spiral arrangement would be displaced horizontally, with a truly radial orientation at
the east-west poles. These reference axes within the
artery wall are shown in Figure 4.
Computer transformations of the results were
made to correct for the angle at which the vessel was
sectioned24 and also for the angular position of each
reading around the circumference of the section.
Results were plotted on the Lambert projection both
in point and in contoured form. We used Fisher
statistics32 to analyze the three-dimensional orientation results. The parameters obtained are the mean
orientation vector; a precision parameter 095 that
defines the accuracy of that mean; and a circular
standard deviation (CSD), which is a measure of the
scatter about the mean orientation. The value of 095
684
Arteriosclerosis and Thrombosis
Vol 11, No 3 May/June 1991
adventitia
media
subendothelium
adventitia
media
subendothelium
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100/ym
FIGURE 2. Polarized-light photomicrographs of segments of two artery walls. Left panel: Vessel No. 29, vertebral artery, pressure
fixed at 30 mm Hg, from a 58-year-old woman. Medial layer shows strong birefringence, while two distinctly separate orientations
can be seen in subendothelial layer. Right panel: Vessel No. 33, basilar artery pressure fixed at 200 mm Hg, from a 68-year-old
woman. In this vessel, subendothelial tissue appears darker, similar to the darker layer in left panel, which is characteristic of a more
longitudinal orientation. Bar=100 yjn.
is the solid angle about the mean direction within
which there is a 95% probability of the presence of
the true mean. The CSD is analogous to the standard
deviation in a Gaussian distribution and is denned as
the solid angle about the mean orientation that
encloses 63% of the data.
Repeatability of Measurements
We set out to test the repeatability of measurement for birefringent tissue oriented at angles clearly
Vessel #29
Media
outside of the plane of section because we learned
that much of the fabric of subendothelial muscle was
not circumferentially aligned and the previous assessment of precision had been done for circumferential
fabric elements relatively close to the plane of
section. For this part of the study, the positions of
measurements were marked on a photomicrograph,
and a series of readings of subendothelial orientation was obtained. The slide was removed and
subsequently repositioned on the stage to permit a
Subendothelium
Outer
Inner
FIGURE 3.
adventitia
media
subendothelium
Vessel # 4
Lambert projections of me-
dial and subendothelial smooth muscle
from two vessels, #29 fixed at 30 mm Hg
and #4 fixed at 200 mm Hg. Point at
center represents a single circumferential
measurement, with degree of helical orientation dependent on the distance toward
north or south pole; degree of spiral orientation is indicated by distance toward east
or west pole. R, radial; C, circumferential;
L, longitudinal.-
Finlay et al
TABLE 1.
Small Area Comparison
SE width
Vessel
NoVsection
6 outer
30
40
70
140
No. of
readings
5
mid
5
inner
12 outer
5
mid
5
inner
29 outer
5
5
mid
5
inner
31 outer
5
mid
5
inner
5
5
5
Helical pitch
(degrees)
-52.2
-47.4
-47.2
-32.0
-27.2
-26.6
+48.6
+31.0
+23.2
+50.6
+40.6
+27.8
Brain Artery Subendothelial Orientation
SD
3.3
1.5
3.1
6.4
55
9.0
4.2
4.3
4.0
4.2
3.5
5.8
Spiral angle
(degrees)
-9.6
-11.2
-8.0
+5.4
+4.6
-2.8
-3.4
-4.4
+3.2
-6.2
-3.0
-0.6
685
SD
3.6
2.4
2.0
2.1
2.7
2.6
5.1
2.9
5.1
2.3
0.7
2.7
Each set of five readings is from a concentric array, arranged in three rows across the vessel wall. "Inner" is the set
closest to the lumen, and "outer" is closest to the media.
SE, subendothelium.
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further series of measurements at the same relocated points. Three complete artery sections were
examined for repeatability (pressure distended at
30, 110, and 200 mm Hg, respectively, at the time of
fixation), which revealed that the average magnitude of the difference between pairs of readings was
5.4±2.8° (mean±SD) for N=60.
Measurements From a Limited Area
Four separate areas from different vessels were
examined in detail to test the variation in alignment
from fiber to fiber. Sets of readings were made from
regions of subendothelium covering the width, which
varied from 30 to 140 /im, and a length of approximately 100 £im. A matrix of three circumferential
rows of five points was marked on the photomicrographs, with the readings between 10 and 50 /xm
apart depending on the width of the subendothelium.
Results from each vessel showed an increasing
trend to a more longitudinal orientation with increasing proportional radial distance from the inner
(next to the lumen) to the outer (adjacent to the
internal elastic lamina) regions of subendothelium
(Table 1). In each of these vessels, only one helical
direction was detected at the region examined,
although in the two wider vessels, there was a
FIGURE 4. Schematic of artery segment, illustrating circumferential (C), longitudinal (L), and radial (R) reference axes.
F, line segment of measured birefringent fiber; 8n helical pitch;
8& spiral angle.
greater difference in angle between the inner and
outer readings. Our attention was drawn to the low
value of SD for each cluster of five measurements
(mean SD of 4.6°, with a range from 1.5° to 9° for
helical pitch, and a mean SD of 2.9°, with a range
from 0.7° to 5.1° for the spiral angle).
Results
Medial Layer at a Range of Pressures
The tunica muscularis has become the alignment
reference for other measurements of artery wall structure obtained by polarized-light microscopy,24 but we
needed confirmation that this layer remained a reliable reference at all fixation pressures. This part of the
study included sections that had been pressurefixedat
four different transmural pressures. For every vessel
examined, the helical and spiral components of alignment were calculated in addition to the precision
parameter a^ and the CSD. As an example, the
results from seven sections, each from an arterial
segment pressure fixed at 60 mm Hg, are shown in
Table 2. Note the average angle of opposing pairs of
helical fibers, which reveal a balance of right- and
left-handed helical organization of medial muscle
fibers. Table 3 compares the average results from all
vessels grouped according to the fixation pressures.
We had anticipated that there might be less coherent alignment of the smooth muscle at low pressures,
which would be revealed by higher values for CSD.
The data did not bear this out. In fact, the striking
feature that has become a hallmark of the muscle
fabric of the tunica media is the extremely narrow
variation in alignment, averaging about 5°, which in
this study has been shown to persist over the entire
possible range of physiological pressure.
Subendothelial Layer at a Range of Pressures
In contrast to the highly coherent smooth muscle
of the tunica media, tissue within the subendothe-
686
Arteriosclerosis and Thrombosis
TABLE 2.
Vol 11, No 3 May/June 1991
Medial Layer Results of Seven Arteries Fixed at 60 mm Hg
Gender/age
(yr)/artery
M63 Vert
M63Bas
M59 Vert
M42MCA
M42 Sup cer
M42 MCA
F85 Ant cer
Mean
No. of
readings
Vessel
No.
16
Helical pitch
(degrees)
-0.4
-0.7
+0.8
+2.0
-2.0
+0.3
-0.8
35
44
17
18
19
33
34
27
29
25
32
20
21
22
Spiral angle
(degrees)
-1.2
-1.0
+ 1.2
+2.2
+ 1.6
+3.6
+0.4
1.0
0.7
SD
CSD
1.3
1.0
1.3
1.2
1.8
1.3
1.2
1.3
0.2
1.6
1.0
4.2
3.8
4.3
3.9
5.2
4.0
3.3
4.1
0.6
Mean values of helical and spiral angles are calculated from numerical mean of each vessel, not taking into account
the sign of the angle or number of readings.
a^ is a measure of precision of the mean value.
CSD, circular standard deviation; M, male; F, female; Vert, vertebral artery; Bas, basilar artery, MCA, middle
cerebral artery, Sup cer, superior cerebellar artery; Ant cer, anterior cerebral artery.
Downloaded from http://atvb.ahajournals.org/ by guest on June 16, 2017
Hum showed considerable organizational variability,
both within individual sections and from vessel to
vessel. Examples of the results from individual sections are shown in Table 4. These data include the
two sections for which Lambert projections have
been shown in Figure 3 (vessel No. 4 fixed at 200 mm
Hg and vessel No. 29 fixed at 30 mm Hg) and the
complete set of results from arteries pressure fixed at
30 mm Hg. Vessel No. 29 has a comparatively thick
subendothelial layer in which two distinct sublayers
could be identified; each layer, when analyzed, was
found to consist of both right- and left-handed helically aligned groups of fibers. Thus, the results for
this section separate into four distinct orientation
groups. Other vessels with a less well developed
subendothelium (i.e., vessel No. 4 in Table 4) do not
have this distinct separation of layers, so only one
helical pair of orientations has been identified.
Table 5 shows the statistical measurements from
the subendothelial layers listed according to pressure
of fixation. We noted that our reference statistical
parameter used to report the dispersion of alignment
about the mean orientation, that is, the CSD, had
widely varying values from vessel to vessel. We interpreted the lack of correlation between CSD and the
pressure of fixation to have resulted from the high
degree of variability for the subendothelium.
From the vessels whose readings could be separated into inner (adjacent to the lumen) and outer
TABLE 3.
Pressure
(mm Hg)
200
110
60
30
(adjacent to the internal elastic lamina) subendothelium, mean values of orientation within these groups
were calculated. These results are shown in Table 6,
with the focus on average helical angle, comparing
inner and outer helical angle at each pressure. For
some vessels, the whole circumference of the subendothelium was subdivided into inner and outer layers, and in others, only certain regions of the artery
wall revealed these two concentric sets of oriented
muscle fibers. The Lambert projection of vessel No.
29 in Figure 3 shows the results clustering toward the
north-south poles in the outer layer, which indicates
a more nearly longitudinal orientation.
Discussion
The directional organization that has been revealed by our own and other studies of the artery
wall33 has led us to seek out what degree of directional order may exist within the subendothelium and
to assess the effect of distending pressure on that
order. The general microscopic appearance is that
the subendothelium is much less precisely aligned
than is the tunica media. In many brain arteries,
especially the smaller ones, it may be thin or nonexistent, whereas in larger vessels, a continuous and
substantial subendothelial layer may be present with
a nonuniform thickness around the circumference,
often with an irregular subdivision of the thicker
regions as revealed with cross polars on the polariz-
Combined Medial Results at Different Pressures
No. of
vessels
6
11
7
9
Mean
No. of
readings
33
36
32
33
Mean helical
pitch (degrees)
SDof
each
set
Mean spiral
angle (degrees)
SD
09!
0.6
1.3
1.0
0.4
0.4
0.9
0.7
0.4
0.7
0.9
1.6
0.9
0.8
1.0
1.0
0.9
1.4
2.4
1.3
1.2
Mean
Mean
CSD
4.3
7.7
4.1
3.8
Mean values of helical and spiral angles at each pressure were calculated from vessel mean results, not from the total
of individual measurements. See footnote to Table 2. as» is a measure of precision of the mean value.
CSD, circular standard deviation.
Finlay et al Brain Artery Subendothelial Orientation
687
TABLE 4. Examples of Subendothelial Layer Results
Pressure
(mm Hg)
200
30
Gender/age
(yr)/artery
Vessel
No.
M70
Vert
4
F58
Vert
29 outer
29 inner
30
30
F58
Bas
F58
Vert
28
30 outer
inner
30
M84
Bas
27 outer
inner
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30
M59
Vert
31 outer
inner
30
F85
Bas
26
No. of
readings
Helical pitch
(degrees)
15
17
26
10
-36.5
+39.4
20
14
22
34
18
13
16
18
17
22
29
8
11
17
13
15
12
26
Spiral angle
(degrees)
-0.8
+0.7
-7.9
-9.3
-57.7
+61.6
-28.0
+28.0
-53.8
+48.8
-29.0
+26.9
+0.5
-5.5
+5.7
+0.6
+2.5
-6.4
-6.1
-13.5
-6.7
-1.6
+0.3
-57.8
+50.7
-47.6
+39.5
-54.6
+53.1
9.9
5.2
11.7
15.7
21.2
9.5
7.9
15.1
16.6
18.2
18.0
21.5
15.2
6.7
6.3
7.4
14.5
14.6
16.4
5.6
4.8
6.8
3.5
4.2
14.5
14.4
9.9
6.2
8.3
7.2
5.6
-18.7
-5.8
+5.9
-5.7
CSD
4.1
5.6
13.1
6.2
+0.5
-2.6
+ 7.1
+32.9
-49.6
+54.6
-53.8
+49.4
-19.2
<*»
6.5
7.1
8.1
7.3
9.5
12.6
14.7
14.9
20.3
In some vessels, subendothelium was divided into inner (next to the lumen) and outer regions, aM is a measure of
precision of the mean value.
CSD, circular standard deviation; Vert, vertebral artery; Bas, basilar artery.
ing-light microscope. From the histological appearance, the subendothelium falls into three broad
categories: 1) vessels with a continuous and thin
uniform subendothelium, with a width as much as
approximately 30% of the medial width; 2) a uniformly thickened subendothelium of thickness similar to that of the tunica media; and 3) the nonuniformly thickened subendothelium with substantial
regions of thickness extending around a portion of
the inner perimeter of the wall. These thickened
atherosclerotic regions were in some instances further subdivided into pathogenic intimal thickening or
intimal pads close to bifurcations.
The majority of the thinner layers were divided
into two separate portions of the perimeter in which
orientations of either positive or negative pitch were
measured. Where these two regions came together
and overlapped, both directions of pitch were re-
corded. The relative proportion of the two main
opposing areas was approximately equal. We are
uncertain about the continuity of the helical groups
of fibers along their length. The thicker endothelial
areas showed a more complex fabric. A few regions
(i.e., Figure 2, left panel) demonstrated a distinct
separation into inner and outer layers from which
two sets of readings could be made, although in many
other areas (i.e., Figure 2, right panel), there was not
a clear distinction between the inner and outer
regions. Some areas around the perimeter showed
the same general helical direction through the subendothelial wall, but with greater pitch angle closer
to the internal elastic lamina, while other areas
revealed two opposing pitch directions. The areas of
atherosclerotic thickening appeared as a continuation of the normal subendothelial layer, although
comparisons of trichrome-stained sections revealed a
TABLE 5. Combined Subendothelial Results at Different Pressures
Pressure
(mm Hg)
200
110
60
30
No. of
vessels
Mean No.
of readings
Mean helical
pitch (degrees)
6
6
5
6
13
22
19
40.3
50.3
51.6
44.8
18
SD
Mean spiral
angle (degrees)
15.9
8.3
11.8
16.3
12.0
12.8
9.6
5.6
SD
7.2
8.9
10.8
4.6
Mean
o»
Mean
CSD
7.7
13.3
15.0
14.7
15.2
6.6
7.5
6.9
Mean angles were calculated from each subgroup (i.e., left- and right-handed helical group), not as mean from
pooled measurements. Two longitudinal groups were excluded. 095 is a measure of precision of the mean value.
CSD, circular standard deviation.
688
Arteriosclerosis and Thrombosis
TABLE 6.
Pressure
(mm Hg)
200
110
60
30
Vol 11, No 3 May/June 1991
Comparison of Inner and Outer Sobendothelial Layers
No. of
vessels
Inner helical
pitch (degrees)
Outer helical
pitch (degrees)
Difference
(degrees)
4
4
40.0
38.3
52.5
52.2
2
29.4
6
34.5
64.6
56.6
12.5
14.2
35.2
22.1
Values of pitch are taken as mean of numerical values, not taking into account the sign. Inner pitch is that closer to
the lumen, and outer pitch is closer to the medial layer.
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smaller smooth muscle volume fraction, and with
silver stain, birefringence in this area was usually
weak. The remaining fabric was found to consist of
collagen, lipid, ground substance, and cell debris.15
Collagen and ground substance are birefringent, but
the ground substance was mostly eliminated by the
alcohol stages of tissue processing,34 which would
account for the weaker birefringence. The fabric in
these regions of substantial thickening was more
variable in organization and in cell content than that
in the normal subendothelial layer. In all vessels from
which we took readings, there were regions in which
it was not possible to make orientation measurements. Two contributing factors were an absence of
birefringent tissue in that area and a lack of coherence of the birefringent components within the small
zone of measurement.
With increasing pressure at time of fixation, we
anticipated a trend to more circumferentially directed subendothelial muscle tissue along with a
reduced scatter about the mean orientation of medial
and subendothelial muscle. The magnitude of circumferential strain that we estimated for a luminal
pressure change from 30 to 200 mm Hg was 35%,
based on the mechanical studies of Scott et al35 for
the larger human brain arteries. Hence, as an example, a muscle layer organized helically at 45° at 30
mm Hg distending pressure would become less helically oriented, to an angle of 36.5° at 200 mm Hg.
This would cause a measurable difference for any
helically organized layer that was consistently present
among all vessels. However, the highly variable direction of organization that we have observed for the
subendothelium indicates clearly that there is no
consistent direction of organization for any of the
regions, thus masking any change that would occur
within individual arteries.
Three consistent findings have been drawn from
the results in this study. 1) Whenever distinct layers
could be identified in the subendothelium, the muscle more adjacent to the arterial lumen was more
circumferential in organization than layers closer to
the tunica media (e.g., 60° for the outer layer compared with the inner muscle at a 30° helical angle for
vessel No. 29 in Table 4, and similarly in Tables 1 and
6). Although still lacking an explanation, these quantitative observations are consistent with other studies
(work on intimal thickening in the human aorta by
Geer,36 on induced atherosclerosis in the rabbit
carotid artery by Hoff and Gottlob,37 and on hyper-
plasia within the subendothelium with atherosclerosis and aging by Buck38). Smooth muscle cells are
dynamic in their organization as illustrated by their
infiltration and proliferation in the subendothelium
with atherosclerosis, aging, or wall injury.16-17 When
grown in culture, smooth muscle cells orient themselves in small colonies or fasciculi of parallel cells,
with random overall orientations of these colonies.7'38
However, as they proliferate in the subendothelial
layer, their orientation is far from random and may
be influenced by pulsating circumferential strain,
with evidence for this proposal coming from studies
of the growth of fibroblasts on cyclically stretched
substrates.39 That is, the cells most recently arrived
from the tunica media by infiltration through fenestrations within the internal elastic lamina would be
oriented longitudinally because of cyclic circumferential strain, but possibly with time, these cells would
become more circumferentially aligned to participate
mechanically. 2) When the helical components were
identified as left- or right-handed helixes, in general
there was a balance of orientation, signaling to us a
preservation of mechanical symmetry (e.g., helical
angle data pairs in Table 4: -57.7° and +61.6°, or
-28.0° and +32.9°). To graphically illustrate this
finding, we plotted the pairs of helical angles for each
layer measured (Figure 5). For symmetrically
matched layers, data would fall on the line of identity, and it can be seen from the figure that the data
do cluster around that line, while spanning angles of
orientation from 20° to 70°. Mechanical symmetry has
been reported for canine elastic arteries,40 and it may
be that the symmetry of organization of the subendothelium is additional evidence of a mechanical
function for that acquired layer of brain arteries in
humans. 3) The scatter about the mean orientation
for both medial smooth muscle and subendothelium
was unaltered by fixation pressure, a finding similar
to observations of hamster cheek arterioles by
Walmsley et al.41 Our explanation is that the cells
themselves have become elongated passively with the
increasing circumferential strain with pressure and
have thus preserved their relative directional organization among neighboring cells. This agrees with the
results of Mulvany and Halpern,42 who studied small,
actively constricted resistance vessels.
We had opportunities to examine locally thickened
areas of brain artery. The sections we examined
varied, from those with very little (<10 iim) subendothelium around the vessel circumference to those
Finlay et al
Brain Artery Subendothelial Orientation
689
80
60
FIGURE 5. Scatterplot of left- versus right-handed helical
angles of pairs of fiber groups in subendothelial layers, with
each sublayer comprising a pair of left- and right-oriented
helixes. Vertical bar represents the average a^ of these two
helical fiber groups.
40
\
20
i
•
30 mm Hg
•
60 mm Hg
O 120mmHg
A 200mmHg
Downloaded from http://atvb.ahajournals.org/ by guest on June 16, 2017
0
20
40
60
RIGHT HELIX
80
with a continuously thickened layer as much as 100%
of the medial width, with some regions of intimal
thickening as much as 300% of the medial width. In
two sections (both fixed at a distending pressure of
200 mm Hg), the smooth muscle was longitudinally
oriented (to within 8° of perfectly longitudinal). In
three other sections, the smooth muscle in the thickened section was aligned more circumferentially than
was the remaining subendothelial layer. In some
cases, the thickened areas were close to bifurcations
and may have been intimal pads. Others may have
been caused by injury to the endothelium or by a
localized atherosclerotic lesion. Clearly, there was
preservation of structure within thickened regions,
but there was a wider range of direction to the groups
of muscle cells when compared with the more common continuous thin subendothelium.
There is a possibility that a primary subendothelial
layer is laid down, due to smooth muscle migration
through fenestrations in the internal elastic lamina,13
with a mainly longitudinal orientation35 associated by
contact guidance and cyclical stretching of the vessel
by pulsation of blood pressure in vivo.39 After migration to the subendothelial layer, the smooth muscle
may become primarily a synthesizing cell for connective tissue,43 generating type HI collagen in the early
atherosclerotic process and subsequently type I collagen.44 As atherosclerosis proceeds, there is a loss of
distensibility of the thickened artery, which is reflected in the variability of orientations of the smooth
muscle fabric. These orientations may vary with the
type of atherosclerosis, with the thickness of the
lesion, and with the position with respect to bifurcations. The nearness to bifurcations is significant
because branch sites show less mechanical strain with
static distending pressure than do straight regions of
brain arteries.45
The mechanisms of dilation and contraction of
arteries are complex, involving many neural vasodilating factors reaching the medial smooth muscle via
perivascular innervation that
directly affects only the
outer layer of the media46 and also involving an
endothelium-dependent relaxing factor released by
endothelial cells in response to blood-borne stimulants.47-48 It is not established whether a thickened
subendothelial layer will inhibit transference of this
relaxing factor, or how much the smooth muscle of
the subendothelium may be involved in the dilation
process. Regulation of vascular tone is achieved by an
equilibrium of all these factors, vasospasm being one
possible effect of an imbalance. We have found a
definite structural symmetry of the smooth muscle
within the subendothelium, comprising regions of
approximately equal and opposite helical arrangements. There are two possible explanations for this.
One reason is that the symmetry might be imposed by
external dynamic or mechanical factors. This would
appear likely to explain an order or alignment, but
there would be no requirement for the balance in
magnitude of the two opposing helixes that we find.
The indication therefore is that the smooth muscle in
that layer has a mechanical and not just a synthesis
role. If this is the case, the significance of that
mechanical function to vasospasm and atherosclerosis has yet to be assessed.
Acknowledgments
The authors would like to thank Robert Goyer,
Chairman, Peter Munavish, and Steve Stewart of
the Department of Pathology for assistance in
obtaining tissue, and Barbara Anderson for preparation of the manuscript.
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Fabric organization of the subendothelium of the human brain artery by polarized-light
microscopy.
H M Finlay, J G Dixon and P B Canham
Arterioscler Thromb Vasc Biol. 1991;11:681-690
doi: 10.1161/01.ATV.11.3.681
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