Kurume Medical Journal, 49,177-183, 2002 Original Article

Regional Diminution of von Willebrand Factor Expression on the Endothelial Covering Arachnoid Granulations of Human, Monkey and Dog Brain

KEISUKE OHTA, TETSUO INOKUCHI, YUUHO HAYASHIDA, TETSUYA MIZUKAMI, TOMOHIRO YOSHIDA AND TARO KAWAHARA

Department of Anatomy and Histology, Kurume University School of Medicine, Kurume 830-0011, Japan

Summary: Arachnoid granulation is a protrusion of the arachnoid membrane into the cranial sinus, and is thought to play an essential role in the (CSF) absorption. Because the cells covering the apex region of the arachnoid granulation have different morphological features compared to the ordinary endothelial cells lining of the cranial sinus lumen, it has been expected these covering endothelial cells perform some specific function in the CSF absorption mechanism. However, little is known about functional differences between the covering endothelium of the arachnoid granulation and the ordinary sinus endothelium. In the present study, the characteristics of the covering cells located at the apex of arachnoid granulations of human, monkey and dog brain were examined by histochemical and immunohistochemical methods. The endothelial cells lining the cranial sinus lumen generally expressed such proteins as von Willebrand factor (vWF), CD31 and glycoproteins containing GS-1 or LE-1 lectin reacting sugar residue which are endothelial cell markers. However, the endothelial cells specifically located at the apex of arachnoid granulations failed to show vWF immunoreactivity, whereas the other endothelial markers were positive in each species we examined. Double staining of vWF antibody with other markers has clearly demon- strated that the endothelial cells on the apex region of arachnoid granulations exhibit no expression of vWF whereas cells lining the lateral region of arachnoid granulations and the luminal surface of ordinary cranial sinuses showed co-localization of these markers. The structural and histochemical differences between endothelial cells located at the apex region of arachnoid granulations and those of the sinus wall may reflect functional differences.

Key words immunohistochemistry, lectin, endothelial marker, arachnoid granulation, von Willebrand factor, endothelial cell, cranial sinus

Although the size and the structure of arachnoid INTRODUCTION granulations are considerably differ considerably by The protrusions of arachnoidmembrane into the cra- species or by age of subject, they are generally nial sinus lumen through the dural wall are called thought to play an essential role in CSF transport arachnoid granulations or arachnoid villi. Since from subarachnoid space to the cranial sinus lumen. Weed's classic work [1] describing the role of arach- These granulations are special bulgings of the inner noid villi in the drainage mechanism of cerebrospinal wall of the cranial sinus, and their surfaces are con- fluid (CSF), many physiological and morphological sidered to be covered with a continuous sheet of studies on arachnoid granulation of many laboratory endothelium [7-9]. A number of studies were focused animals [2-4] and humans [5,6] have been reported. on the mechanisms of CSF drainage across the sinus

Received for publication August 5, 2002 Corresponding to: Dr. Keisuke Ohta, Department of Anatomy and Histology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. Tel: +81-942-31-7541 Fax: +81-942-31-7555 E-mail: [email protected] 178 OHTA ET AL.

endothelial covering. Ultrastructual features of the thiopental anesthesia (25 mg/kg), and the animals cells covering the apical regions of arachnoid granu- were perfused via the aorta with physiological saline, lations are known to be quite different from those of followed by 4% paraformaldehyde/0.1 M cacodylate endothelial cells located at the surface of the ordinary buffered solution. The arachnoid granulations with inner wall of sinuses: The superficial covering cells surrounding were then dissected out from of the apex of arachnoid granulations have many the superior saggital sinus, and further fixed in the giant intracellular vacuoles which are thought to same fixatives for 3 hrs at 4•Ž. provide a channel for bulk flow of the CSF from the subarachnoid space into the cranial sinus lumen Immunohistochemistry and lectin-histochemistry [10,11 ]. Although many detailed observations of The tissues were rinsed in 0.1 M cacodylate ultrastructural characteristics of the covering cells buffer, and incubated in 15% sucrose solution in have been reported, the histochemical features of buffer overnight at 4•Ž. They were then embedded these cells has remained largely unknown. Therefore, in OCT-compound (Tissue-Tec; Miles Laboratories in the present study, we investigated the histochemi- Inc., Elkhart, Ind., USA) and were frozen in chilled cal differences between the covering cells of arach- ethanol by dry ice. noid granulations and those of the ordinary sinus The antibodies against vWF and CD31, and the walls. Immunohistochemical and lectin-histochemi- lectins of Lycopersicon esculentum (LE) and cal studies using four commonly-used endothelial Griffonia simplicifolia I (GS-1), were used as the markers (von Willebrand factor (vWF), CD31 anti- endothelial cell markers. Seven micrometer frozen bodies, GS-1 and LE-1 lectins) revealed that the sections on slide-glasses were applied to hema- arachnoid granulations are distinctly covered with toxylin-eosin (HE) staining and also to the histo- endothelial cell lining, and that cells in the apical chemistry. Several sections of the arachnoid granula- region of arachnoid granulations show no or only tions were treated with 1% bovine serum albumin weak expression of vWF. A similar mode of vWF (BSA) and 2.5% normal goat serum in 0.01 M phos- expression was reported in human arachnoid granula- phate buffered saline (PBS) and subsequently incu- tions by Kida et al. who interpreted their finding as a bated in diluted primary antibody or biotinylated proof of a lack of endothelial lining [6]. We used lectin. The best staining contrast was obtained at a dual immunohistochemical staining methods with the dilution of 1:1600 for rabbit anti-human vWF anti- antibodies against vWF and other endothelial marker body (DAKO, Denmark), 1:50 for mouse anti-human proteins or sugar residues in several animals and also CD31 antibody (DAKO, Denmark), 1:1600 for in human beings. We have clarified that the defect of biotinylated-LE (Sigma Chemical Co., St. Louis, vWF expression in the apex region of arachnoid USA) and 1:1600 for biotinylated-GS-1 (Vector, granulations indicates some region-specific charac- Burlingame, Ca., USA). The specimens for immuno- teristic of the endothelium but not a lack of endothe- histochemical studies were subsequently reacted with lial covering itself. the secondary antibodies, biotinylated anti-rabbit or

anti-mouse IgG. The biotin-marked antigens or sugar

residues were visualized by immuno-peroxidase MATERIALS AND METHODS methods (Vectastain ABC Kit; Vector, Burlingame,

Human arachnoid granulations were obtained Ca., USA) or by immuno-fluorescence methods

from ten cadavers during regular educational dissec- (Amersham, Great Britain). tions for medical students at Kurume University For double fluorescence histochemistry of vWF

School of Medicine. Arachnoid granulations which and other markers, the sections were incubated

were dissected out from cadavers were re-fixed overnight with anti-vWF antibody and the other

by 4% paraformaldehyde/0.1 M cacodylate buffer endothelial marker antibody or lectin at the concen-

(pH 7.3) for 4 hrs at 4•Ž. trations described above. Following incubation with

Arachnoid granulations of laboratory animals primary antibodies or lectins, the sections were were obtained from three adult Japanese monkeys reacted with biotinylated anti-mouse IgG (Vector,

(Macacus fuscatus) and two canines. The mainte- Burlingame, Ca., USA), if necessary, followed by nance of the animals and all experimental procedures fluorescein linked anti-rabbit IgG (Amersham, Great

were carried out in accordance with the Guide for Britain) and Texas Red-labeled streptoavidin. The

Animal Experimentation, Kurume University School double-stained sections were observed by confocal

of Medicine. All surgeries were performed under laser scanning microscope (Carl Zeiss, LSM-310).

Kurume Medical Journal Vol. 49, No. 4, 2002 VWF FXPRESSION IN ARACHNOID GRANULATION 179

The specificitics of primary antibodies were checked is surrounded by fibrous capsule. The surface of the in sections reacted without the antibodies or Iectins. fibrous capsule of the arachnoid granulations in the Those studies revealed the absence of cross-fractions present study was seen to be covered with endothe- with other primary or secondary antibodies. lial sheets, although these were hard to identify in the light microscopic sections stained with hematoxylin Scinning electron mcroseope and eosin (Fig. la). Canine arachnoid granulations were observed On the other hand, the "fibrous capsule". which under the scanning elect on microscope (SEM). The was commonly observed in human arachnoid gran- specimens dissected out as above were fixed by 2% ulations, was not observed in any arachnoid granula- paraformaldehyde + 2.5% glutaraldchyde solution in tions of monkeys and dogs. The apex of the granula- 0.1 M cacodylate buffer (pH 7.3) for 2 hrs at room tions was frequently lobulated with rugged surfaces temperature. Specimens were rinsed in the buffered (Figs 1b and 2a). and the inner structure of the granu- solution containing 7% sucrose buffer for one day, lations was composed of loose arrangements of the and post-fixed in 2% OsO4 (pH 7.4) for 2 hrs. They arachnoid tissue. U ltrastructural observation of the were then dehydrated through a graded series of dog arachnoid granulation using SEM showed that acetone. were transferred to isoamvl acetate and were the coverings of the rugged surface of the granulations dried at critical point using liquid CO2. The dried was consisted of spherical and/or slender-shaped specimens were coated with gold in an ion-sputter cells (Fig. 2b). Although these cells were different coater and were observed using a HITACHI S-800 from ordinary endothelial cells. the spherical-slender SEM. cell layer seemed to be continuous with the usual endothelial covering of the venous sinus wall sur- rounding the arachnoid granulations. RESULTS Thus, the usual histological observations alone There were demonstrated the arachnoid granula- do not sufficiently, characterize and discriminate the tion in all animal and human samples observed in arachnoidal covering cells. Therefore, in this study. this study, but their size and shapes were various in the arachnoid granulations were further investigated each species (Fig. 1). Human arachnoid granulations by immuno-histochemical and Icctin-histochemical mostly had a "central core" structure as described methods (Table 1). The ordinary endothelial cells previously by Kida et al [6]. The "central core" covering the venous sinus walls of the humans and according to their descriptions is a network of arach- the two kinds of animals showed strong binding noid cells intermingled with collagenous fibers. and activities to LE lectin and vWF antibody. Further.

Fig. 1. Light photomici raph of as scrtion of huIfan (a) and monke, (b), arachnoid granulations (AG) herniatine from the tiubarachnoid space (SAS) into the superior saggital sinus (SSS) through the dural wall (D).

Kurume Medical Journal No.4,2002Vol.49, 180 OHTA ET AL

Fig. 2. Scanning electron-mieroacopic observation of clog arachnoid granulation in the superior saggital sinus. In this low magnitude photomicrograph (a), arachnoid granulations protrude towards the Sinus lumen. The apical region of the arachnoid erunulation is shown at higher magnification (b).

TABLE 1. Immuno-histochemical and lectin-histochemical staining of endothelial cell in arachnoid g ranulations and surrounding ducal sinus surface hr endothelial cell markers

Intensity of the reactivity on the endothelial cells sere scored: •¬, very strong reaction: •¬

, strong reaction: +, scene reaction: -, negatis e reaction.

concerning the sinus wall endothelium. CD31 anti- and GS-1 Icctin (in dog) revealed the same type of body was only detected in human beings and mon- positive reactions on the surface cell layer of the keys but not in dogs. Also, GS-1 was only found on :uachnoid granulations same as those of the ordinary the clog endothelium in. but not in the others (Table sinus wall (Fig. 3a. Table 1 ). Since these marker sub- 1). stances were constantly and continuously detected on In the arachnoid granulation area. CD31 antibody the surface of the arachnoid granulations, it was esti- (in human and monkey). LE Icctin (in all species). mated that the surface of the arachnoid granulation

Kurume Medical Journal V ol.49, No 4, 2002 VWF EXPRESSION IN ARACHNOID GRANULATION 181

Fig. 3. Immunohistochcmical staining of mon- key arachnoid granulations for CD31 (a) and von Willebrand factor (b). CD31 immunoreactis ities are observed throughout the endothelial cell linings on the venous sinus wall and on the surface of arachnoid granulations (arrow). On the other hand, von Willebrand factor immunarcactivitics are seen along the surface of dura (d) (arrowhead). hut not on the arachnoid granulation (arrow). SSS: superior saggital sinus; SAS: subarachnoid space

Fig. 4. Laser scanning microscopic micrographs of arach- noid granulation of human (a), monkey (b) and dog (c). Dual immunolocalization of von Willebrand factor (green) and

CD31 (a, b) or LE lectin (c) (red) are confirmed only in the endothelial cells located at the lateral and/or neck region

(arrowheads) of arachnoid granulations and in ordinary sinus wall (arrows). Bar=250ƒÊm (a). 50ƒÊm (h) or 100ƒÊm (c) .

Kurume Medical Journal Vol. 49, No. 4, 2002 182 OHTA ET AL. was completely covered with endothelial cells con- human arachnoid granulations have confirmed that secutively. However, immunohistochemistry on the fetal and child arachnoid villi are distinctly covered vWF demonstrated negative, or weak and discon- with sinus endothelium [5,15], and our double stain- tinuous immunoreactive products in the arachnoid ing studies also show that the apical region of granu- granulations of all species we examined (Fig. 3b lations are covered with cell linings which express arrow, Table 1). some endothelial markers other than vWF. Although The double staining studies using antibodies the possibility remains that some giant arachnoid against vWF antibody and other marker substances granulations in human being occasionally lose the in order to establish the precise localization of vWF, endothelial investment at the apex region of due to CD31(human and monkey) and LE lectin (dog) have some type of mechanical forces, our findings indicate clearly shown that, in the adluminal sinus wall, the presence of an endothelial covering in most of granulated vWF immunoreactivity was the same as animal arachnoid granulations, and in humans as that observed in most other surfaces. This was the well. Therefore, the special mode of distribution of case with the other markers as well. Nevertheless the vWF suggests their functional difference, not the vWF immunoreactivity was negative or weak and absence of endothelial cells. discontinuous in the surface of arachnoid granula- The vWF is known to play an important role in tions (Fig. 4). These features were found in the speci- platelet adhesion and aggregation mechanisms and to mens obtained not only from human beings (Fig. 4a), localize within Weibel-Palade bodies which are seen but also from monkeys (Fig. 4b) and dogs (Fig. 4c). in the cytoplasm of vascular endothelial cells [16- 18]. A scarcity of Weibel-Palade granules has been reported in vascular capillaries [19,20], as well as in DISCUSSION differentiating endothelial cells during angiogenesis Arachnoid granulations are protrusions of arach- in various tissues [21]. Therefore, the absence of noid membrane into the cranial sinus which are vWF expression dose not indicate the absence of thought to play an essential role in CSF absorption. endothelium, but only suggests a regional, functional Since the structures of arachnoid granulations are specificity of endothelial cells which, in those areas, known to vary greatly from species to species, we do not need vWF. need to compare the histochemical features of arach- The precise mechanism of the CSF drainage noid endothelial linings, and consider whether a through the endothelial covering remains to be finding is species specific or universal. In this study, defined. Since the arachnoid granulation is supposed histochemical observations of the monkey, dog and to provide a main route for drainage of cerebrospinal human arachnoid granulations demonstrate that the fluid from the subarachnoid space to the venous endothelial cell linings of the apical region of the sinus, there is no doubt that the endothelial covering granulations have smaller amounts of vWF than the of the arachnoid granulation must be closely ordinary sinus endothelium. Since the endothelial connected with the CSF drainage phenomenon. cell of each kind of tissue was detected by several Considering humoral drainage through endothelial endothelial markers even in the apex of arachnoid sheets, a close similarity between the humoral trans- granulations, the granulations of the present three fer in Schlemm's canal of the eye and that of arach- species, at least, are considered to be covered with noid granulation of the brain is noted. The endothe- endothelial cell linings, completely. lial cells lining one side of Schlemm's canal wall The present histochemical observations are in possess giant vacuolar structures that are thought to good agreement with a majority of previous studies act in the outflow system of fluids from the anterior on the structure and functions of arachnoid granula- ocular chamber [10]. The endothelial vacuolations tions in laboratory animals, describing that the seen in the Schlemm's canal and arachnoid granu- surface of the arachnoid granulations are completely lation (Fig. 2) [11,22], may be related to the bulk covered by endothelial cell linings [7-9,12]. However, flow of large amount of humors. Thus, these spe- some authors have reported that the human arachnoid cialized endothelial cells may be expected to provide granulation in their ultrastructural and immunohisto- some regionally specific function, different from chemical study, are only partially covered by usual endothelial cells. Although these cells must endothelial cells and were much more regularly have some special biochemical characteristic related invested with the arachnoid cell layer (mesothelium) to the CSF drainage, little is known so far. The pres- [6,13,14]. Observations of the development of ent results demonstrated the presence of endothelial

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