Okajimas Folia Anat. Jpn., 58(4-6)

17 Pages·2012·3.94 MB·English

Okajimas Folia Anat. Jpn., 58(4-6) : 819-836, March 1982
Are the Arachnoid Villi Really the Main Drainage Route
for the Cerebrospinal Fluid into the Blood Stream ?
An Electron Microscopic Study
By
PAULO H. HASHIMOTO, TAKAHIRO GOTOW,
TAKAO ICHIMURA and TOMIO ARIKUNI
Department of Anatomy, Osaka University Medical School,
Osaka 530, Japan
-Received for Publication December 1, 1981-
Key Words : Arachnoid villi, Circumventricular organs, Fine structure, Cerebrospinal
fluid drainage, Blood-brain barrier
Summary : The arachnoid granulations or villi are not always present in lower
mammals and even in human late prenatal fetus. They grow in number and size
with advanced age, and thus it seems appropriate to regard them as safety-valves
for the cerebrospinal fluid (CSF) to be prevented from its hypertension accompanied
by the age and by the increasing volume of the brain. Using 2 macaque monkeys
and 12 rats, and by means of scanning and transmission elecron microscopies, tracer
experiments with horseradish peroxidase, elastic staining of thin sections, and
freeze-substitution of rapidly frozen brains, evidences are presented to show that
the drainage sites of the CSF in the brain are the sites being devoid of the blood-
brain barrier, that is, the subfornical organ at the root of the choroid plexus of
lateral ventricle, the pineal body at the root of the choroid plexus of third ventricle,
and the area postrema at the root of the choroid plexus of fourth ventricle, and
also the hypophysis and its vicinity, and the choroid plexus itself.
Since Weed (1923) proposed the arach- pressure conditions in the arachnoid villus
noid villi as the site of absorption of of the monkey. Welch and Friedman
CSF into the venous system, it has been (1960), Jayatilaka (1965), Gomez and Potts
believed that the CSF drains into the (1974), as well as Julow, Ishii and Iwa-
superior sagittal sinus through them, buchi (1979) reported existence of valves
although the arachnoid villi or granula- or channels within the arachnoid villus
tions appear later than does the CSF, although Shabo and Maxwell (1968) denied.
both ontogenetically and phylogeneticaliy any direct connection between subara-
(Osaka et al., 1980). Tripathi (1977) chnoid CSF in the villi and venous blood
showed vacuoles which opened to the in the sinus. Hashimoto and Hama (1968),
sagittal sinus, and disappeared under low showed that an intravascularly injected
Supported in part by grants from the Ministry of Education of Japan.
819
820 P. H. Hashimoto, T. Gotow, T. Ichimura and T. Arikuni
horseradish peroxidase (HRP) stained the microscope.
area postrema and the choroid plexus,
which are supplied by fenestrated capil- Tracer experiment with intraventricular
lanes and consequently devoid of the HRP
blood-brain barrier. In the present study Four rats were injected with 20 mg
we will show some electron microscopic HRP. (type VI, Sigma Chemical Co.)
evidence that brain regions which are dissolved in 0.1 ml saline or artificial CSF
devoid of the blood-brain barrier function (van Deurs et al., 1978) into the right
as routine sites of CSF drainage, and lateral ventricle. Ten to 15 min. after
the arachnoid villi may act as safety- the end of a slow tracer infusion the
valves against high pressure conditions, brains were fixed by perfusion with 1%
or emergency exits of the CSF.
glutaraldehyde +1 % paraformaldehyde
in 0.1 M cacodylate bufler pH 7.3, and
Materials and methods processed for benzidine reaction (Graham
and Karnovsky, 1966) as described in
Scanning electron microscopy followed by detail in a previous paper (Gotow and
thin section observations Hashimoto, 1979).
Heads of macaque monkeys (a Macaca
fuscata and a Macaca mulatta) were Elastic fiber staining for electron micros-
fixed by vascular perfusion through as- copy
cending aorta with buffer-diluted aldehyde Four rats were used. Thin sections
mixture (Karnovsky, 1965). The falx were cut from blocks of brains fixed by
cerebri was dissected out, and the sagittal perfusion as usual. They were first
sinus was opened by the sagittal bisection. stained with 0.6% orcein in 70% ethanol
Pieces of tissues were cut and immersed for 15 min. in room temperature, and
in a solution containing 2% sucrose, 2% successively rinsed in 95% ethanol con-
sodium glutamate, and 2% glycine for taining 3.4 ml 1 N HC1 in each 100 ml,
2 hr., then in 2% tannic acid for 2 hr. then in 70% ethanol without HCI, and
(Murakami, 1973). After washing in then in water, and dried in air for 30
distilled water for 1 hr., the tissue blocks min. They were then stained with 2%
were postfixed in 2% osmium tetroxide uranyl acetate in 50% ethanol for 15 min.
for 2 hr. The blocks are dehydrated in and with lead citrate for 10 min. (Naka-
acetone, immersed in isoamyl acetate, mura et al., 1977).
and dried at the critical point with liqui-
fled carbon dioxide. They are then Rapid freezing and freeze-substitution
pasted on sample holders, coated slightly Thin and small piece of tissue was
with gold, and observed in a Hitachi taken from living brain by a fresh razor
S-700 scanning electron microscope blade, put on an Eiko RF-2 divice (Hi-
equiped with a field emission gun. tachi Ltd.), and immediately touched onto
After the SEM observation, tissue blocks a pure copper block cooled to —196°C
containing arachnoid villi were re-im- with liquid nitrogen. The frozen tissue
mersed in acetone and in propylene oxide, was then thrown into 4% 0s04 in
and embedded in Epon. Thin sections dry ice-cold acetone and kept overnight
were cut on a Sorval MT-I or -II micro- at —78°C. The tissue in acetone osmium
tome, stained with aqueous uranyl acetate were brought to —20°C and kept for
and with lead citrate, and observed under 2 hr., then at 4°C for 2 hr., and finally
a Hitachi HU-11A or -11E electron at room temperature for 2 hr. They
Circumventricular Organs as Sites of CSF Drainage 821
were washed in acetone, and in ethanol, pineal body (Fig. 7), area postreme (Gotow
and stained en bloc with 3% uranyl and Hashimoto, 1979), and choroid plexus,
acetate in ethanol, washed in ethanol, 10 to 15 min. after the end of slow
immersed in propylene oxide, and embed- administration for about 20 min. Similar
ded in Epon. phenomenon was also observed in the
ventral aspect of the diencephalon : the
Results hypophysis, the median eminence, and
the lamina terminalis.
Arachnoid villi of the monkey
Elastic fibers connecting both basal
Macroscopical arachnoid granulations
laminae of the endothelium and of the
were difficult to detect along the interior
of the sagittal sinus of the young adult perivascular astrocytic processes were
detected in the perivascular space of ca-
animals. Several arachnoid villi were
observed by the scanning microscope at pillaries of the subfornical organ, the
branching sites of the sinus vein (Fig. 1). pineal body, and the area postrema (Fig.
8). They were also found in the pia
The endothelium of the sinus reflects
mater of these organs (Fig. 8). Detailed
to cover the surface of villi where it
three dimensional fine structure of the
makes deep and shallow folds. No appar-
ent endothelial fenestrae were observed pars fiamentosa and the pars amorpha of
elastic fibers in these circumventricular
although microvillous protrusions were
organs will be described elsewhere (Ichi-
present (Fig. 2). Thin membranous por-
mura and Hashimoto, in preparation).
tions of the endothelium were observed
Non-ciliated and flattened specialized
as dark ovoid flecks on the scanning
ependyma of the subfornical organ is
micrograph. No mesothelial lining is
transformed into the choroid epithelium
present under the endothelium of ara-
(Fig. 9) just same as the case in the
chnoid villi which shows folds but is
area postrema (Gotow and Hashimoto,
nevertheless continuous (Fig. 3). The
1979).
framework of the villus is composed
of bundles of collagen fibrils, fibroblasts
Intercellular space of the freeze-substitut-
and lymphocytes. Huge vacuoles were
ed fiber bundles
often obsered in the endothelial cells
Afer freeze-substitution, tissue space
(Figs. 3, 4). Judging from the detached
of the white matter with interwoven
basal lamina, the cytoplasmic attenuation
neuronal processes exhibited approxima-
in figure 4 does not look natural. They
tely three times larger than that in case
are probably corresponding to the dark
of chemical fixation (Fig. 10, Text-Fig. 1).
ovoid flecks of the scanning electron mi-
The fornix for the subfornical organ
crographs. Huge opening of the inter-
cellar cleft between two tight junctions (Fig. 9), the stria medullaris for the
at the bottom of the fold (Figs. 3, 5) pineal body, and the fasciculus gracilis
for the area postrema may provide a
looks also unna tural. A better preserved
tissue spacechannel within the central
villus endo- thelium looks more natural
nervous system as a route for CSF ab-
with perfect tight junctions and well
sorption (Text-Fig. 2).
attached basal lamina (Fig. 6).
Circumventricular organs of the rat Discussion
Intraventricularly injected HRP was
detected in the lumen of fenestrated ve- We have shown some fine structural
nous capillaries of the subfornical organ, evidence of CSF drainage from circum-
822 P. H. Hashimoto, T. Gotow, T. Ichimura and T. Arikuni
ventricular organs in addition to scanning
compression against perivascular CSF
pressure.
Hirokawa and Kirino (1980) showed the
real existence of inercellular space in the
central nervous system by a rapid freez-
ing method. Nabeshima et al. (1975)
found the meningeal CSF barrier at the
uppermost arachnoid barrier layer, and
no barrier at the pia mater nor the mar-
ginal glial layer. Nakayama (1976) demon-
stratedan opening of the central canal in
the film terminale internum.
Because the appearance of arachnoid
villi or granulations delays ontogenetically
Space B = 1.43 and phylogenetically as compared to that
of choroid plexus (Osaka et al., 1980), it
Space C/13 = 2.33 is not reasonable to consider that the
Space C/A 3.35 formers are essential for CSF absorption.
They seem more suitable to be considered
as safety-valves for CST to be prevented
from being hypertensive.
Text-Fig. 1. A comparison between
simple calculations of the volume of inter- It is natural, in the sense of the pri-
cellular spaces in 3 model cases. C resem- mary meaning, that the choroid plexus
bles Fig. 10. of the lateral ventricle is provided with
the subfornical organ as the site of CSF
drain, the choroid plexus of the third
and transmission electron microscope ob-
ventricle with the pineal body, and the
servations of a continuous but easy to
choroid plexus of the fourth ventricle
break endothelial covering of the ara-
with the area postrema, in addition to
chnoid villus. The valvular opening of
the lamina terminalis, the median emi-
the arachnoid endothelium (Tripathi,
nence and the hypophysis. The fornix,
1977) looks like a broken outflow of a
the stria medullaris, the fasciculus gracilis
vacuole such as in our figure 4. Also the
and many other tracts and fasciculi may
tubular channel in the core (Jayatilaka,
1965) looks like a broken opening of a play the role of interstitial CSF pathways.
Even without other circumventricular
vacuole such as in our figure 5.
organs, the total volume of choroidal ven-
Cserr et al. (1977) suggeted the flow
ous capillary beds seems enough to absorb
of cerebral interstitial fluid to drain into
the whole CSF produced by their own
the fenestrated vessels of the choroid
choroid plexus, which have well develo-
plexus and other periventricular areas.
We demononstrated it electron micros- ped apical tight junctions to prevent direct
invasion of intraventricular CSF to the
copically (Gotow and Hashimoto, 1979).
interstitium (Gotow and Hashimoto, 1979)
Elastic fibers connecting basal laminae of
even though a transepithelial transport
the fenestrated endothelium and of the
has also been reported (van Deurs et al.,
perivascular astrocytic processes (Hashi-
1978).
moto, Gotow and Ichimura, 1981) may
well prevent the venous capillary from Veins from most of organs mentioned
Circumventricular Organs as Sites of CSF Drainage 823
Text-Fig. 2. A schematical drawing to show the new concept
of CSF circulation. Thin arrow : flow of intraventricular CSF. Thick
arrow : flow of subarachnoid CSF.
above join into the vena cerebri magna, as indicated by removal of extracellular
and then the sinus rectus which joins markers from rat caudate nucleus. Exp.
the sagittal sinus to make the transvese Eye Res., 25 (Suppl.) : 461-473, 1977.
sinus ; thus this could be misunderstood 2) Gomez, D. G. and Potts, D. G.: The
that the drainage occurred via arachnoid surface characteristics of arachnoid
granulations. Arch. Neurol., 31 : 88-93,
1974.
3) Gotow, T. and Hashimoto, P. H.: Fine
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824 P. H. Hashimoto, T. Gotow, T. Ichimura and T. Arikuni
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Circumventricular Organs as Sites of CSF Drainage 825
PLATES
826 P. H. Hashimoto, T. Gotow, T. Ichimura and T. Arikuni
Explanation of Figures
Plate I
Fig. 1. Scanning electron micrograph of an arachnoid villus of a macaque monkey viewed from
the luminal side of the sinus sagittalis. The venous endothelium recurves to continue to
curves the endothelial lining of the villus at star. x 190.
Fig. 2. A higher magnification of Fig. 1. Many infoldings, dark ovoid flecks, and microvillous
protrusions are visible. Except for few caveolae, no fenestrae are seen. An equivalent
section level similar to that of Fig. 3. is indicated by the white line. x 950.
Fig. 3. A conventional thin section electron micrograph of a monkey arachnoid villus taken
from the same block as has been observed by the scanning microscope. The endothelial
lining is continuous, but it contains several large vacuoles (arrows) , and seems ready to
be broken at the rectangular fields which are shown at higher magnifications in Figs. 4,
5. Collagen fibrils, fibroblasts and lymphocytes are visible in the core of the villus..
x 3,500.
827
Plate I
Paulo H. Hashimoto, et al.
828 P. H. Hashimoto, T. Gotow, , T. Ichimura and T. Arikuni
Plate II
Fig. 4. An endothelial cell between two junctions becomes attenuated, of which the basal
lamina (B) is detached between arrows. Gold coating is indicated by the open arrow.
x 23,000.
Fig. 5. The endothelial intercellular space is extensively widened between apical and besal
tight junctions (arrows) . B, basal lamina. x 23,000.
Fig. 6. Covering endothelium of the arachnoid villus is generally continuous with well developed
tight junctions (arrow) . Basal lamina (B) is well underlined. No fenestra and few
micropinocytotic vesicles are observed. Gold coating is indicated by the open arrow.
x 26,000.