Download Elastic Elements in the Media and Adventitia of Human

Elastic Elements in the Media and Adventitia
of Human Intracranial Extracerebral Arteries
F. T.
M £ R E I , M.D.,
329
F. GALLYAS, P H . D . ,
A N D Z. H O R V A T H ,
M.D.
SUMMARY We find that the media and adrentitia of adult human cerebral arteries contain elastic fibers
forming a dense, coherent network, similar to that found in muscular arteries of the same size in other organs.
The external elastic layer in the adult human is masked for the currently employed staining methods. By treatment with 90% formic acid before fixation, the original staining character of elastic tissue can be restored. The
light microscopic and scanning electron microscopic features of this network of elastic fibers are presented.
Stroke, Vol 11, No 4, 1980
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THE ELASTIC MATERIAL in intracranial extracerebral arteries is almost all concentrated in the internal elastic layer, in contrast to the muscular arteries
of other organs, whose media and adventitia contain
abundant elastic fibers in an outer layer.1 The susceptibility of the cerebral arteries to injury, as well as the
frequent occurrence of aneurysms in these vessels have
been attributed partly to this peculiar distribution of
the elastic material.*"'
In the arteries of the circle of Willis and its large
branches at birth, Hassler and Larsson' found a few
medial and adventitial elasticfibers.Their number per
unit volume increased up to the age of 2 years. The
fibers formed a clearly defined external elastic layer in
20% of the children examined, only an incoherent
loose "pile" in 70%, and scarcely any in 10% over
each of the intimal cushions encountered. The density
of the external elastic fibers appeared to decrease
markedly with increasing age. Only their remnants
were found in about 10% of the adults above 30 years
of age, mainly in the vertebral and basilar arteries,
and at times, over atheromatous areas in the more distal arterial branches. However, Wolff" has called
attention to the fact that the meningeal arteries facing
the cranium contain elastic elements in their media
and adventitia throughout life. Crompton4 pointed out
that lack of an external elastic layer is restricted to the
arteries of the adult human brain, and the intracranial extracerebral arteries in various animals have
a well developed network of elastic fibers located
mainly in the boundary zone between the media and
adventitia. All these statements are based on the
observation of histologic preparations fixed, embedded and stained in the usual manner.
In a study of agents used for the isolation of elastin'
on non-fixed blood vessels, we found that the media
and adventitia in the arteries of the adult human brain
do contain elastic elements in large amounts.
From the Department of Neurosurgery, University of Pecs
Medical School, Ifjusag-utja 31, 7624 Pecs, Hungary.
Supported by the Scientific Research Council, Ministry of
Health, Hungary /3-21-0301-04-O/M/.
Material and Methods
The circle of Willis and its main branches, together
with short sections of their cortical branches, were dissected before fixation from the base of the brain in 40
unselected patients at autopsy. Their ages ranged from
7 to 80 years with 5 collected for each decade. Arteries
with atheromatous changes visible to the naked eye
were not studied.
The following portions of the arteries were dissected
and reserved for the usual histologic procedures: 1) the
right internal carotid artery with its bifurcation into
the anterior and middle cerebral arteries, 2) the right
middle cerebral artery and its main branches, 3) the
right anterior cerebral artery and the origin of its first
main branch, 4) the right posterior cerebral artery
together with the posterior communicating artery, 5)
the right vertebral artery, and 6) a 20 mm long segment of the basilar artery.
The remaining areas were subjected to one of the
following procedures capable of dissolving all components of the vessel wall except the elastic fibers and
membranes which are resistant to extreme chemical
and physical measures: 1) treatment with 0.1 N
sodium hydroxide at boiling point for 30 min;7 2) incubation with 90% formic acid at 45°C for 48 h;s 3)
enzymatic digestion.* The remnants were washed with
3 changes of distilled water for about 10 min each.
Despite the laceration and crushing during these
procedures, the original arrangement of the arteries
was easily recognized, and short segments which
suffered no damage were excised. These arterial
segments were incised longitudinally using an
operating microscope and microsurgical instruments.
The external elastic layer was separated from the internal elastic lamina and then air-dried on glass slides
in an unfolded state. Some of the preparations were
processed for scanning electronmicroscopy, and
others were stained by a silver technique developed by
one of us.10 To avoid mechanical damage some
arterial segments were mounted without incising. In
such preparations both the internal and the external
elastic laminae could be studied under the microscope.
Five mm lengths were cut from the arterial
segments reserved for common histologic procedures,
then fixed in 4% formalin for one day, embedded in
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paraffin, cut at 6 //m and finally stained by one of the
following methods: 1) resorcin-fuchsin, 2) orcein, 3)
Verhoeff's hematoxylin method, 4) toluidine blue,
topooptical staining,11 5) silver techniques for the
demonstration of elastic elements10 and smooth muscle cells.12 The remaining arterial segments were exposed to 90% formic acid at room temperature for 24
hours, before fixation and subsequent processing.
Digestion by formic acid disorganizes the structure of
collagen and smooth muscle cells without totally
removing them from the arterial wall ("partlydigested" materials). Although the arteries shrink
markedly under the effect of formic acid, making the
internal elastic lamina fold to an unusual extent, the
main layers of the blood vessels (intima, media,
adventitia) can easily be recognized.
The main arteries of the brain from 10 more
patients at autopsy were incubated in 90% formic acid
at 45 °C for 48 h without having been cut into
segments. The external elastic layer was separated
from the internal elastic lamina and the intimal elastic
layer. The pooled external elastic material was
purified, then processed for amino acid analysis as
described by Ross and Bornstein.14
Results
The procedures used for the elimination of
"unwanted" components of the arterial wall were all
VOL 11, No 4, JULY-AUGUST
1980
equally effective in isolating elastic fibers and membranes. After digestion by any of them, 3 concentric
layers of elastic elements were detected in each of the
arterial segments examined, independently of their
origin and age (fig. 1). After being cut open longitudinally, the outer layer became separated from the
adjoining one without any external force. The middle
layer looked like a thick, bright, gleaming sheet under
the operating microscope and could be identified as
the internal elastic lamina, based on its fenestration.13
For this reason, the inner layer must have been
situated in the intima and the outer layer in the media
and/or the adventitia of the arterial wall. The outer
layer appeared under the operating microscope as an
opalescent, loose sheet. This could be stretched
repeatedly to about twice its original length without
apparent damage to its structure.
The light and scanning electronmicroscopic images
of the totally digested arterial segments dried onto
glass plates revealed that the outer layer consisted of a
dense network offibers(fig. 2). The thicker fibers had,
on their surface, furrows running parallel to their
longitudinal axes, as if they were made up of several
thin fibers cemented together (fig. 3). The density of
the network of external elastic fibers decreased
gradually in the more distal part of the artery and only
a few fibers could be found in the branches smaller
than 0.5 mm (fig. 4). The external elastic layer is composed of fairly long fibers merging into each other at
FIGURE 1. Operating microscopic view of the elastic sheets remaining in the stump of the
middle cerebral artery of an adult human after removal of other tissue components by means of
digestion by 90% formic acid at 45° C for 48 hours, a) elastic elements of the intima
("Begleitmembran"), b) internal elastic lamina, c) external elastic layer.
ELASTIC FIBERS IN CEREBRAL ARTERIES/Mere/ et al.
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FIGURE 2. External elastic network in the basilar artery of an adult human, isolated by formic acid. A ir-driedpreparations, a) stained by silver" for light microscopy, X40; (b-d) scanning
electronmicroscopic pictures, XI000, X3000, XI0,000.
their endings, constituting a coherent network with
relatively few branching points (fig. 5). "Free" fiber
endings were observed in a negligible number.
In the media and adventitia of the non-digested
arterial segments obtained from 32 of our 40 patients,
the staining methods and silver techniques rendered
visible only sporadic elastic fibers (fig. 6a). In the
remaining 8 patient samples a weakly developed external elastic layer was stained, mainly in the vertebral,
basilar and internal carotid arteries. In contrast, a
dense elastic network was demonstrated in the media
and adventitia in each of the partly-digested arterial
segments of the circle of Willis and its main afferents
and efferents (fig. 6b). The density of fibers gradually
decreased with the diameter of the artery (fig. 6c, d).
In general, the media contained fewer but thicker
fibers than the adventitia, where they were located
mainly in the vicinity of its inner boundary. There
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FIGURE 3. High power scanning electronmicroscopic image of the preparation in Fig. 2. Note
the furrows on the surface of the fibers running parallel to their longitudinal axes.
X30.000
were considerable individual variations in this pattern,
e.g., 1) only the media contained elastic fibers; 2) the
adventitial fibers were thicker and 3) were located in
the outermost zone of the adventitia. The number of
patients was too small for any conclusion to be drawn
concerning the significance of the individual
variations. No direct connection was found between
the internal elastic lamina and the external elastic
layer, but a small number of fibers connecting the
medial and the adventitial elastic network was
observed.
Each of the staining methods visualized the external
elastic fibers in the partly digested as well as in the
totally digested preparations. The silver technique
demonstrating smooth muscle cells in non-digested
materials12 proved to be the most suitable for this purpose. Even short treatment (partial digestion) by formic acid abolishes the capacity of the smooth muscle
cells to react with silver ions under the circumstances
required by the staining technique in question and
enhances that of the elastic elements.10
The amino acid composition of the external elastic
layer of the cerebral arteries (table) was similar to that
of the ligamentum nuchae,14 young and old aorta, and
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ELASTIC FIBERS IN CEREBRAL ARTERIES/A/erw et al.
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FIGURE 4. External elastic network in the a) internal carotid artery, bj middle cerebral artery,
c) first main branch of the middle cerebral artery, d) in a thick cortical branch of the latter;
isolated by formic acid and dried onto glass plates without the internal elastic lamina having
been removed. The density of the external elastic layer decreases in a distal direction. X250.
TABLE Amino Acid Composition of the External Elastic Elements of Human Cerebral Arteries Compared with
that of Other Elastic Tissues and Collagen
Amino adds
Glycine
Alanine
Valine
Leucine
Isoleucine
Phenylalanine
Methionine
Glutamic acid
Aspartic acid
Proline
Hydroxyproline
Arginine
Lymne
Tyrosine
External
elastio layer
El as tin
of human
from bovine
cerebral
liga men turn
arteries
nuchae1*
Residues per 100 residues
27.1
34.8
11.2
5.3
1.6
1.8
0.2
1.0
1.0
5.3
1.0
1.0
9.8
1.5
EUutin
from human
Elutin
Elutin
pulmonary
from young11
from old 1
artery"
humin aorta
human aorta *
gN per lOOgN
32.4
22.3
13.5
6.1
2.6
3.1
—
26.1
23.2
13.0
4.5
2.1
1.7
0.1
1.5
0.6
12.0
1.1
5.4
0.7
0.7
0.4
10.1
—
1.8
0.5
1.4
1.8
21.3
21.6
11.5
4.8
2.3
2.0
0.4
3.0
1.1
9.2
—
4.3
1.2
1.8
32.0
—
12.9
4.9
2.3
—
—
1.8
0.6
11.0
—
—
—
—
Cowhide
oollagen
26.2
9.5
3.4
3.2
2.3
4.2
0.8
11.3
6.3
15.1
14.0
8.8
4.5
1.4
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FIGURE 5. External elastic network in the anterior cerebral artery, isolated by formic acid,
then dried on a glass slide in a moderately stretched state. The fibers are not uniform in
thickness. Several branching points but no free fiber ending can be seen. X10,000
pulmonary artery15 in so far as it contained more than
80% non-polar amino acids and less than 2% hydroxyproline. In contrast, collagen can be characterized by
a hydroxyproline content of about 14% and a relatively
high proportion of other amino acids with charged
side chains.16 The external elastic layer of the cerebral
arteries was rich in lysine. A low content of lysine is
regarded as characteristic of cross-linked/non-soluble/elastin.17
Discussion
Our findings obtained from arteries digested by
various biochemical procedures for the isolation of
elastic material suggest strongly that the arteries of
the adult human brain are similar to other muscular
arteries of comparable caliber in their content of
elastic structures in the media and adventitia. Comparison of the non-digested preparations with the
ELASTIC FIBERS IN CEREBRAL ARTERIES/Mere/ el al.
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FIGURE 6. a) Anterior cerebral artery of an adult human, stained by silver10 without having
been digested; b) an adjacent segment of the same material, stained by silver12 after partial
digestion by formic acid; c and dj thin cortical branches protruding from the posterior cerebral
artery of an adult human, stained by silver12 after partial digestion by formic acid. The partial
digestion by formic acid unmasks the external elastic fibers of the adult human intracranial
arteries. Only the thinnest cortical branches lack elastic fibers in the media and adventitia
farrows).
partly digested ones indicates that the elastic fibers
constituting these elastic structures are masked for the
available staining methods, unlike the elastic fibers of
the muscular arteries of other organs. The "masking"
may consist of in vivo blocking of certain reactive
groups in the molecular structure of the elastic tissue,
which may play a role in the stainability of the latter.
The treatment of non-fixed cerebral arteries with formic acid reverses this effect and restores the original
staining character of the elastic fibers. Attempts to
reproduce this effect of formic acid in paraffin sections
of formol-fixed materials failed. The resistance of the
external elastic layer of the cerebral arteries to the
isolation procedures used, as well as their high-grade
extensibility after the isolation, make it possible to
conclude that the "masking" is not accompanied by a
major transformation of the chemical composition
and structure of the elastic material.
The chemical nature of a number of tissues,
traditionally called "elastic" by histologists, such as
the elastic membranes and fibers in intimal cushions
and arteriosclerotic lesions, the internal elastic lamina
of veins and small arteries, etc. has been questioned
repeatedly by several workers,18"22 many of whom
produced histochemical evidence indicating that these
tissues were composed of a special kind of collagen —
? type III collagen. It was resistant to the procedures
for isolating elastin and appeared as a homogenous
mass under the transmission electron microscope. It
could be demonstrated by both the so-called elastic
staining methods and various staining and histochemical methods believed to be specific for collagen. Such
tissues were termed by Wolff18 "pseudoelastica." To
avoid ambiguity, Puchtler et al.22 introduced the following terminology: Elastin fibers or Membranes for
structures composed of true elastin; Pseudo Elastica
in the above sense; and Elastic Fibers and Membranes
for structures composed of either true or pseudo elastin. This terminology is used throughout the present
paper. The amino acid composition of the external
elastic fibers of the cerebral arteries resembles that of
elastin obtained from ligamentum nuchae or elastic
arteries rather than that of collagen (see table), but
this observation does not allow differentiation of its
elastin or pseudo elastin nature.
The scanning electronmicroscopic findings on the
structure and branching pattern of the fibers making
up the external elastic layer of the cerebral arteries
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is similar to findings obtained in ligamentum nuchae.23> 24~28 This supports the somewhat older theory
of Romhanyi,27 formed from polarization microscopic
observations that elastic fibers are composed of
parallel subfibers cemented together.
Why are the external elastic fibers of the cerebral
arteries invisible by the currently employed staining
methods? Hassler and Larsson3 and Crompton
assumed that the external elastic fibers of muscular
arteries served to dampen the pulse. These fibers seem
to appear when they are needed, such as after birth,
but disappear gradually, becoming nonfunctional, i.e.
the "lack" of elastic fibers over intimal cushions. The
skull of the adult was regarded by those authors3-4 as
a closed box full of water, which prevents any dilatation of a rubber tube running through it. The outer
elastic layer begins to "disappear" in human cerebral
arteries at the age of coalescence of the bones of the
skull. The skulls of animals cannot function as a
closed box because the total surface area of the
foramina for vessels, nerves and spinal cord is
relatively large when compared with the internal surface area of the skull. Using this theory, it could be
assumed that a special physiological process takes
place in the external elastic fibers which have become
dysfunctional, and this results in an alteration of their
chemical composition. The latter might manifest itself
in a change of the staining properties of elastin.
Acknowledgment
The authors are indebted to Dr. Gy. Soltesz, Department of
Pediatrics, University of Pecs Medical School for performing the
amino acid analysis.
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Elastic elements in the media and adventitia of human intracranial extracerebral arteries.
F T Mérei, F Gallyas and Z Horváth
Stroke. 1980;11:329-336
doi: 10.1161/01.STR.11.4.329
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