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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 References structure of the ependyma and intercellular junctions in the area pos- 1) Cserr, H. F., Cooper, D. N. and Milhorat, trema of the rat. Cell Tissue Res., 201 : T. H.: Flow of cerebral interstitial fluid 107-225, 1979. 824 P. H. Hashimoto, T. Gotow, T. Ichimura and T. Arikuni 4) Graham, R. C. and Karnovsky, M. J.: in the meninges and marginal glia. J. The early stages of absorption of injected Comp. Neur., 164: 127-170, 1975. horseradish peroxidase in the proximal 13) Nakamura, H., Kanai, C. and Mizuhira, tubules of mouse kidney : ultastructural V.: An electron stain for elastic fibers cytochemistry by a new technique. J. using orcein. J. Histochem. Cytochem., Histochem. Cytochem., 14: 291-302, 1966. 25: 306-308, 1977. 5) Hashimoto, P. H., Gotow, T. and 14) Nakayama, Y.: The openings of the Ichimura, T.: On the tissue space in central canal in the film terminale the central nervous system. A contribu- internum of some mammals. J. Neurocy- tion to the CSF absorption. Acta Anat. tol., 5: 531-544, 1976. Nippon., 56 : 303, 1981 (Abstract). 15) Osaka, K., Handa, H., Matsumoto, S. 6) Hashimoto, P. H. and Hama, K.: An and Yasuda, M.: Development of the electron microscope study on protein cerebrospinal fluid pathway in the nor- uptake into the brain regions devoid of mal and abnormal human embryos. the blood-brain barrier. Med. J. Osaka Child's Brain, 6: 26-38, 1980. Univ., 18: 331-346, 1968. 16) Shabo, A. L. and Maxwell, D. S.: The 7) Hirokawa, N. and Kirino, T: An morphology of the arachnoid villi. A ultrastructural study of nerve and glial light and electron microscopic study in cells by freeze-substitution. J. Neurocy- the monkey. J. Neurosurg., 29: 451- tol., 9: 243-254, 1980. 463, 1968. 8) Jayatilaka, A. D. P.: An electron 17) Tripathi, R. C.: The functional mor- microscopic study of sheep arachnoid phology of the outflow system of ocular granulations. J. Anat., 99 : 635-649, 1965. and cerebrospinal fluids. Exp. Eye Res., 9) Julow, J., Ishii, M. and Iwabuchi, T.: 25 (Suppl.) : 65-116, 1977. Arachnoid villi affected by subarachnoid 18) Van Deurs, B., M011er, M. and Amtorp, pressure and haemorrage. Scanning 0.: Uptake of horseradish peroxidase electron microscopic study in the dog. from CSF into the choroid plexus of the Acta Neurochirurg., 51 : 63-72, 1979. rat with special reference to transepi- 10) Karnovsky, M. J.: A formaldehyde- thelial transport. Cell Tissue Res., glutaraldehyde fixative of high osmolality 187: 215-234, 1978. for use in electron microscopy. J. Cell 19) Weed, L. H. The absorption of Biol., 27 : 137A-138A, 1965 (Abstract). cerebrospinal fluid into the venous 11) Murakami, T.: A revised tannin-osmium system. Am. J. Anat., 31: 191-221, method for non-coated scanning electron 1923. microscopic specimens. Arch. histol. 20) Welch, K. and Friedman, V.: The jap., 36: 189-193, 1974. cerebrospinal fluid valves. Brain, 83: 12) Nabeshima, S., Reese, T. S., Landis, D. 454-469, 1960. M. D. and Brightman, M. W.: Junctions 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.

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brain barrier, that is, the subfornical organ at the root of the choroid plexus of . cellar cleft between two tight junctions Nakayama (1976) demon-.
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