Acta Oto-Laryngologica
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Ultrastructure of the Guinea Pig Cochlear Aqueduct: An Electron Microscopic Study of Decalcified Temporal Bones R. Toriya, T. Arima, A. Kuraoka & T. Uemura To cite this article: R. Toriya, T. Arima, A. Kuraoka & T. Uemura (1991) Ultrastructure of the Guinea Pig Cochlear Aqueduct: An Electron Microscopic Study of Decalcified Temporal Bones, Acta Oto-Laryngologica, 111:4, 699-706, DOI: 10.3109/00016489109138402 To link to this article: http://dx.doi.org/10.3109/00016489109138402
Published online: 08 Jul 2009.
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Date: 31 March 2016, At: 19:08
Acta Otolaryngol (Stockh) 1991; 11 1: 699-706
Ultrastructure of the Guinea Pig Cochlear Aqueduct An Electron Microscopic Study of DecalciJed Temporal Bones R. TORIYA,' T. ARIMA,' A. KURAOKA'.' and T. UEMURA' From the Departments of Otolaryngology and Anatomy, Kyushu University, Fukuoka,Japan
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Toriya R, Arima T, Kuraoka A, Uemura T. Ultrastructure of the guinea pig cochlear aqueduct. An electron microscopic study of decalcified temporal bones. Acta Otolaryngol (Stockh) 1991; 11 1: 699-706.
The ultrastructure of the guinea pig cochlear aqueduct was examined using semi-thin and thin sections. The lumen of the cochlear aqueduct was occupied by a sparse meshwork of fibroblasts and delicate connective tissue trabeculae. The periotic tissue lining the bony wall of the aqueduct was composed of multiple layers of both elongated cells and densely arranged laminae of collagen fibrils. These structures were identical to those of the dura mater and the arachnoid. The opening to the perilymphatic space of the scala tympani also contained connective tissue trabeculae, but the arrangement of fibroblasts was more compact here than in the main part of the duct. These structural features suggest that fluid can move freely through cochlear aqueduct, and that the effects of sudden pressure changes in the CSF may be protected against by the densely and perpendicularly arranged fibroblast at the opening to the perilymphatic space. Key words:perilymphatic space, perilymphatic ooze, ultrastructuralstudy, dura and arachnoid rnater.
INTRODUCTION The cochlear aqueduct is a duct which runs between the perilymphatic space of the scala tympani and the subarachnoid space in the temporal bone. It has been suggested (i) to supply the perilymph from the cerebrospinal fluid (CSF) (I-3), (ii) to protect against bacterial and viral infections (4, 5 ) , (iii) to regulate pressure differences between the perilymphatic space and the CSF (6-8),and (iv) to be a potential channel for perilymph oozing in stapes surgery for otosclerosis (9). The morphological features of the cochlear aqueduct have been mainly examined using serial sections of celloidin-mounted specimens in animals (10-13) and humans (14-1 8). In I98 1, Nishimura et al. (2) examined the structure of the guinea pig cochlear aqueduct using scanning electron microscopy. However, the ultrastructure of this aqueduct is not yet well understood. In this study, we used semi-thin and thin sections of epon-mounted, decalcified samples t o investigate the detailed morphological features of the guinea pig cochlear aqueduct using transmission electron microscopy. The function of the cochlear aqueduct is also discussed in this article. MATERIAL AND METHODS
Light and transmission electron microscopy Thirty adult guinea pigs with a normal Preyer's reflex weighing 250-400 g were used in this study. After pentobarbital anesthesia, both temporal bones and the attached dura rnater were gently dissected out, pre-fixed with 3%glutaraldehyde in 0. I M cacodylate buffer for 48 h. and decalcified in 5 % ethylenediaminetetraacetic acid (EDTA) for 7 days. Decalcified samples were post-fixed with 1% osmium tetroxide for 3 h, dehydrated in a graded series of ethanols, and embedded in epon. For light microscopy, semi-thin sections were prepared with a Jeol JUM-7 ultramicrotome and stained with toluidine blue. For transmission electron microsce py, thin sections were prepared. stained with uranyl acetate and lead citrate, and examined under a Jeol 100s electron microscope at 80 kV.
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Scanning electron microscopy Decalcified temporal bones were trimmed under an operating microscope, immersed in 50% dimethylsulfoxide (DMSO), and freezecracked under liquid nitrogen to reveal the interior of the cochlear aqueduct (19). After rinsing with 0.1 M cacodylate buffer, samples were postfixed with 1 % osmium tetroxide for 3 h. The fixed samples were consecutively treated with 2%osmium tetroxide for 2 h, 4%tannic acid for 8 h, and 2 % osmium tetroxide for 2 h. They were then dehydrated in a graded series of ethanols, immersed in t-butyl alcohol for 30 min freeze-dried in an Eiko ID-2 freeze-drying machine (Eiko Engineering, Mito, Japan), sputtercoated with platinum, and examined under a Jeol JSM-840A scanning electron microscope.
RESULTS The mean dimensions of the various parts of theguinea pig cochlear aqueduct were as follows: total length, 21.08 mm (range: 19.5-22.0 mm); maximum diameter of the opening to the subarachnoid space, 3.06 mm (range: 2.8-3.4 mm): a diameter of the narrowest segment, 58 pm (range: 40-80 pm): and the diameter of the opening to the perilymphatic space, 118 pm (range: 100-130 pm). The lumen of the cochlear aqueduct was widely contiguous with the subarachnoid space, contained CSF, and was traversed by delicate connective tissue trabeculae. The bony walls of the cochlear aqueduct were covered by both the dura mater and the arachnoid, which were continuous with the structures surrounding the brain (Fig. 1). The delicate connective tissue trabeculae were composed of blood vessels and fibroblasts, which showed a sheet-like appearance (Fig. 2). At the opening to the perilymphatic space of the scala tympani there was no actual barrier between the perilymphatic space and the Iuman ofthe cochlear aqueduct (Figs. 3 and 4). However, the fibroblasts of the connective tissue trabeculae were arranged more compactly and densely at this site than in the rest of the duct (Fig. 3). While these fibroblasts were arranged parallel to the long axis of the aqueduct in the main duct, they were perpendicular to the long axis at the opening to the perilymphatic space (Figs. 2 and 4). Scanning electron microscopy also detected no barrier at the opening of the cochlear aqueduct to the perilymphatic space (Figs. 5 and 6). There were many cribriform pores at the opening (Fig. 5) and the diameter of such pores was large enough for red blood cells to move freely through them (Fig. 6). Transmission electron microscopy showed the detailed structure of the connective tissue components of the trabeculae and the periotic tissues of the cochlear aqueduct. The connective tissue trabeculae were mainly composed of flattened and elongated fibroblasts and bundles of collagen fibrils. These fibroblasts were in contact with one another and formed a cellular network, while the bundles of collagen fibrils were closely associated with the fibroblasts and supported their network in the lumen of the cochlear aqueduct (Fig. 7). The periotic tissue were composed of multiple layers of both fibroblasts and densely arranged laminae of collagen fibrils (Fig. 8). These multilayered structures corresponded to the dura mater. Several inner layers of the periotic tissues were composed of thin flattened cell processes of spindleshaped cells and had no collagen fibrils between them (Fig. 8), and these layers corresponded to the arachnoid. Contacts between the fibroblasts belonging to the connective tissue trabeculae in the aqueduct lumen were clearly observed in thin sections. At some contact points, the intercellular space was 15-20 nm and was filled with rather electron-dense material, while the adjacent cytoplasm contained dense filamentous material. These structural features correspond to those of an intermediate junction (Fig. 9). Typical gap junctions were also frequently identified as septi-laminar membrane structures (Fig. 10). Many sites of punctate fusion of the plasma membrane were frequently observed between the fibroblasts, and these punctate
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Fig. 1. Low power view of the duct and its opening to the subarachnoid space. The lumen of the cochlear aqueduct is occupied by a sparse meshwork of cells. Note that the glossopharyngealnerve is located at the site of the opening to the subarachnoid space. x70. Fig. 2. The sparse meshwork of delicate connective tissue trabeculae is composed of both flattened fibroblasts and blood vessels. The fibroblasts are longitudinally arranged in the duct lumen. Note that several layers of the flattened cells cover the bony wall. ~ 2 8 0 . Fig. 3. Low power view of the opening to the perilymphatic space of scala tympani (So). The density of the fibroblasts seems to be more compact than in the main duct lumen. Arrows indicate the round window membrane. There is no bamer between the lumen of the cochlear aqueduct and the perilymphatic space (arrowheads). x 70. Fig. 4. No barrier can be seen at the opening to the perilymphatic space (arrowheads).Several layers of fibroblasts are arranged perpendicularly at the opening to the perilymphatic space. ~ 2 8 0 .
fusions usually may correspond to tight junctions with single or several strands (Fig. 10) ( 19-20). DISCUSSION Although the morphological features of the cochlear aqueduct have been investigated by many researchers, most of those studies used 20 to 25 pm serial sections of celloidin-mounted specimens (3, 1&15, 18). Since the mean diameter of the narrowest portion of the guinea pig cochlear aqueduct is 58 pm, 20 to 25 pm appears to be too thick to allow accurate investigation. In this study, we made semi-thin sections of epon-mounted specimens that were 0.2 to 0.5 pm thick for light microscopy. Thus, we could clearly demonstrate a sparse meshwork of fibroblasts and delicate connective tissue trabeculae in the lumen of the cochlear aqueduct.
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Figs. 5 and 6. Scanning electron microscopic view of the opening to the perilymphatic space of the scala tympani. There are many cribriform pores at the opening and these pores are large enough for red blood cells to move freely through them (arrowheuds). x220 and ~ 9 3 0 ,respectively.
These structural features were identical to those of the connective tissue components observed in semi-thin sections of spur resin-mounted subarachnoid tissue from the human brain (21 ). Transmission electron microscopy also showed that the ultrastructure of the delicate connective tissue trabeculae in the cochlear aqueduct was identical to that of the connective tissue of the subarachnoid space in the murine and human brains (20-23). Regarding the opening to the perilymphatic space of the scala tympani, the existence or otherwise of a barrier membrane has been an important problem. While some researchers reported the presence of a barrier membrane between the cochlear aqueduct and the perilymphatic space aAer studying serial sections of celloidin-mounted specimens (1 5 , 18). others could not find any such barrier structure (10, 14). In 1981, Nishimura et al. examined the tine structure of the guinea pig cochlear aqueduct using scanning electron microscopy and reported that there was no barrier membrane between the aqueduct and the perilymphatic space (2). In the present study, we reexamined the tine structure of the cribriform pores at the opening of the cochlear aqueduct to the perilymphatic space using scanning electron microscopy. We demonstrated that the sparse meshwork of fibroblasts and delicate connective tissue trabeculae was present even at the opening to the perilymphatic space, and that there was no specific bamer between the aqueduct and the perilymphatic space. However, we found that the fibroblasts of the connective tissue trabeculae in the cochlear aqueduct were more compact at
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Fig. 7. Transmission electron microscopic view of the meshwork of fibroblasts. Many bundles of collagen fibrils are closely associated with the fibroblasts (urrows). Arrowheads indicate the inner few layers of the periotic tissue which correspond to the arachnoid. x 3 400.
Fig. 8. Transmission electron microscopic view of the periotic tissue lining the bony wall of the cochlear aqueduct. Note the multiple layers composed of flattened fibroblasts and densely arranged laminae of collagen fibrils. Arrowheads indicate the arachnoid. CF: collagen fibrils. x8 200.
the opening to the perilymphatic space than in the rest of the duct. Moreover, the arrangement of the fibroblasts at the opening to the perilymphatic space was perpendicular to the long axis of the aqueduct, whereas the fibroblasts in the main duct were parallel to the long axis. Thus, it is possible that in thick sections, such as serial sections of celloidin-mounted specimens, a pseudomembranous structure may sometimes be observed at the opening of the duct due to the density and the arrangement of the fibroblasts at this site. The periotic tissue lining the bony wall of the cochlear aqueduct has been morphologically examined using light and scanning electron microscopy (2, 3, 10, 11, 14, 16. 18). Although
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Figs. 9 and 10. High magnification view of cell-to-cell contacts in the meshwork of fibroblasts. Fig. 9. Arrows indicate that at some contacts the intercellular space is 15-20 nm and is filled with electrondense material, while the adjacent cytoplasm contains dense filamentous material. This type of contact is an intermediate junction. X36000. Fig. 10. Arrowheadr indicate typical gap junctions. Note that there are many punctate fusions of the plasma membrane, possibly tight junction, between the fibroblasts belonging to the connective tissue trabeculae (arrows). ~84000.
both the dura mater and the arachnoid have been observed to enter the cochlear aqueduct from the brain, the fine structure and the nature of the periotic tissue has not yet been determined using thin sections. In the present study, transmission electron microscopy of thin sections clearly demonstrated the fine morphological features of the periotic tissues. The total length of the periotic tissue, including the opening to the perilymphatic space, was a multilayered structure composed of both elongated cells and densely arranged laminae of collagen fibrils. These structural features of the periotic tissue are identical to those of the dura mater and the arachnoid (2&23). The fine structure of the periotic tissues of the guinea pig vestibular and cochlear perilymphatic spaces has been examined previously using decalcified specimens, and it has been proposed that the tissues lining these spaces were morphologically identical to the arachnoid and lacking a component corresponding to the dura mater (19,24). Thus, it may be concluded that the lumen of the cochlear aqueduct is the same as the subarachnoid space, while both the vestibular and cochlear perilymphatic spaces may correspond to an atypical subarachnoid space. The patency of the cochlear aqueduct has been examined in lower animals by tracer experiments (3, 11) and pressure-monitoring experiments (6-8), and patency has been generally accepted to be present. Our results morphologically support this concept. In addition, the
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high density and the arrangement of the fibroblasts at the opening to the perilymphatic space suggest that the cochlear aqueduct may serve as a protective mechanism against sudden pressure changes in the CSF. Schuknecht & Reisser (9) have proposed that the perilymphatic ooze, which is encountered in stapes surgery for otosclerosis, is partly du to a widely patent cochlear aqueduct. Further experiments studying the ultrastructure of the human cochlear aqueduct are now underway at our laboratory.
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ACKNOWLEDGEMENTS We thank Ms Yuriko Arima for her helpful assistance and Mr T. Kanamaru for photographic help. This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education. Science and Culture (No.02857239).
REFERENCES 1. Ohyama K, Salt AN, Thalmann R. Volume flow rate of perilymph in the guinea pig cochlea. Hear Res 1988; 35: 119-30. 2. Nishimura S, Yanagita N, Inafuku S, Handoh M, Futaysugi Y, Miyake H. A scanning electron microscopic study of the guinea pig cochlear aqueduct. ORL 1981; 43: 79-88. 3. Schuknecht HF, Seifi AE. Experimental observations on the fluid physiology of the inner ear. Ann Otol Rhinol Laryngol 1963; 72: 687-712. 4. Berlow SJ, Caldrarelli DD, Matz GJ, Meyer DH, Harsch GG. Bacterial meningitis and sensonneural hearing loss: A prospective investigation. Laryngoxope 1980; 90:1445-52. 5 . Yanagita N, Koide J, Yokoi H. Morphological change in the cochlear aqueduct following herpes simplex virus inoculation into the subarachnoidal space. Acta Otolaryngol (Stockh) 1988; Suppl456 106-10. 6. Beeutjes BIJ. The cochlear aqueduct and the pressure of cerebrospinal and endolabyrinthine fluids. Acta Otolaryngol (Stockh) 1972; 73: 112-20. 7. Carlborg B, Densert B, Densert 0. Functional patency of the cochlear aqueduct. Ann Otol Rhinol Laryngol 1982; 91: 209-15. 8. Carlborg B, Farmer JC. Transmission of cerebrospinal fluid pressure via the cochlear aqueduct and endolymphatic sac. Am J Otolaryngol 1983; 4 273-82. 9. Schuknecht HF, Reisser C. The morphological basis for perilymphatic gushers and oozers. Adv Otorhinolaryngol 1988; 3 9 1-12. 10. Matsumoto N. A histological study of the cochlear aqueduct. J Otolaryngol Jpn 1952 55: 388-96. 11. Lindsay JR, Schuknecht HF, Nefe WD, Kimura RS. Obliteration of the endolymphatic sac and cochlear aqueduct. Ann Otol Rhinol Laryngol 1952; 61: 697-716. 12. Lindsay JR, Nefe WD, Schuknecht HF, Berlman HB. Obliteration of the ductus endolymphaticus and their accompanying venous channels. Acta Otolaryngol (Stockh) 1953; 43: 176-89. 13. Kimura RS, Schuknecht HF, Ota 01. Blockage of the cochlear aqueduct. Acta Otolaryngol (Stockh) 1974; 77: 1-12. 14. Lempert J, Meltzer PE, Wever EG, Lawrence M, Rambo JHT. Structure and function of the cochlear aqueduct. Arch Otolaryngol 1952; 55: 134-45. 15. Palva T, Dammert K. Human cochlear aqueduct. Acta Otolaryngol (Stockh) 1969; Suppl246: 1-57. 16. Palva T. Cochlear aqueduct in infants. Acta Otolaryngol (Stockh) 1970; 7 0 83-94. 17. Wodyka J. Studies on cochlear aqueduct patency. Ann Otol Rhino1 Laryngol 1978; 87: 22-8. 18. Kelemen G, Fuente ADL, Olivares FP. The cochlear aqueduct: structural considerations. Laryngoscope 1979; 89: 63945. 19. Arima T, Shibata Y,Uemura T. The ultrastructure of the supporting system in the guinea pig semicircular canal. Eur Arch Otolaryngol 1990,247: 25MO. 20. Nabeshima S,Reese TS, Landis DMD, Brightman MW. Junctions in the meninges and marginal glia. J Comp Neurol 1975; I 6 4 127-70. 21. Alcolado R, Weller RO, Pamsh EP, G a d D. The cranial arachnoid and pia mater in man: anatomical and ultrastructural observations. Neuropathol Appl Neurobiol 1988; I 4 1-17. 22. Hutchings M, Weller RO. Anatomical relationships of the pia mater to cerebral blood vessels in man. J Neurosurg 1986; 65: 316-25.
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23. Krisch B, Leonhardt H. Oksche A. Compartments and perivascular arrangement of the meninges covering the cerebral cortex of the rat. Cell Tiss Res 1984 238: 459-74. 24. Franke K. Fine structure of the tissue lining the cochlear perilymphatic space against the bony labyrinthinecapsule. Arch Otolaryngol 1979; 222: 161-7. .Munuscript reeeir?pdOctober 18. 1990: accepted January 8. 1991
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Address for correspondence: Ryuzo Toriya, Department of Otolaryngology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812, Japan
(Slockh)11 1
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Ultrastructure of the Guinea Pig Cochlear Aqueduct: An Electron Microscopic Study of Decalcified Temporal Bones R. Toriya, T. Arima, A. Kuraoka & T. Uemura To cite this article: R. Toriya, T. Arima, A. Kuraoka & T. Uemura (1991) Ultrastructure of the Guinea Pig Cochlear Aqueduct: An Electron Microscopic Study of Decalcified Temporal Bones, Acta Oto-Laryngologica, 111:4, 699-706, DOI: 10.3109/00016489109138402 To link to this article: http://dx.doi.org/10.3109/00016489109138402
Published online: 08 Jul 2009.
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Article views: 13
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Citing articles: 2 View citing articles
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Date: 31 March 2016, At: 19:08
Acta Otolaryngol (Stockh) 1991; 11 1: 699-706
Ultrastructure of the Guinea Pig Cochlear Aqueduct An Electron Microscopic Study of DecalciJed Temporal Bones R. TORIYA,' T. ARIMA,' A. KURAOKA'.' and T. UEMURA' From the Departments of Otolaryngology and Anatomy, Kyushu University, Fukuoka,Japan
Downloaded by [University of Saskatchewan Library] at 19:08 31 March 2016
Toriya R, Arima T, Kuraoka A, Uemura T. Ultrastructure of the guinea pig cochlear aqueduct. An electron microscopic study of decalcified temporal bones. Acta Otolaryngol (Stockh) 1991; 11 1: 699-706.
The ultrastructure of the guinea pig cochlear aqueduct was examined using semi-thin and thin sections. The lumen of the cochlear aqueduct was occupied by a sparse meshwork of fibroblasts and delicate connective tissue trabeculae. The periotic tissue lining the bony wall of the aqueduct was composed of multiple layers of both elongated cells and densely arranged laminae of collagen fibrils. These structures were identical to those of the dura mater and the arachnoid. The opening to the perilymphatic space of the scala tympani also contained connective tissue trabeculae, but the arrangement of fibroblasts was more compact here than in the main part of the duct. These structural features suggest that fluid can move freely through cochlear aqueduct, and that the effects of sudden pressure changes in the CSF may be protected against by the densely and perpendicularly arranged fibroblast at the opening to the perilymphatic space. Key words:perilymphatic space, perilymphatic ooze, ultrastructuralstudy, dura and arachnoid rnater.
INTRODUCTION The cochlear aqueduct is a duct which runs between the perilymphatic space of the scala tympani and the subarachnoid space in the temporal bone. It has been suggested (i) to supply the perilymph from the cerebrospinal fluid (CSF) (I-3), (ii) to protect against bacterial and viral infections (4, 5 ) , (iii) to regulate pressure differences between the perilymphatic space and the CSF (6-8),and (iv) to be a potential channel for perilymph oozing in stapes surgery for otosclerosis (9). The morphological features of the cochlear aqueduct have been mainly examined using serial sections of celloidin-mounted specimens in animals (10-13) and humans (14-1 8). In I98 1, Nishimura et al. (2) examined the structure of the guinea pig cochlear aqueduct using scanning electron microscopy. However, the ultrastructure of this aqueduct is not yet well understood. In this study, we used semi-thin and thin sections of epon-mounted, decalcified samples t o investigate the detailed morphological features of the guinea pig cochlear aqueduct using transmission electron microscopy. The function of the cochlear aqueduct is also discussed in this article. MATERIAL AND METHODS
Light and transmission electron microscopy Thirty adult guinea pigs with a normal Preyer's reflex weighing 250-400 g were used in this study. After pentobarbital anesthesia, both temporal bones and the attached dura rnater were gently dissected out, pre-fixed with 3%glutaraldehyde in 0. I M cacodylate buffer for 48 h. and decalcified in 5 % ethylenediaminetetraacetic acid (EDTA) for 7 days. Decalcified samples were post-fixed with 1% osmium tetroxide for 3 h, dehydrated in a graded series of ethanols, and embedded in epon. For light microscopy, semi-thin sections were prepared with a Jeol JUM-7 ultramicrotome and stained with toluidine blue. For transmission electron microsce py, thin sections were prepared. stained with uranyl acetate and lead citrate, and examined under a Jeol 100s electron microscope at 80 kV.
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Scanning electron microscopy Decalcified temporal bones were trimmed under an operating microscope, immersed in 50% dimethylsulfoxide (DMSO), and freezecracked under liquid nitrogen to reveal the interior of the cochlear aqueduct (19). After rinsing with 0.1 M cacodylate buffer, samples were postfixed with 1 % osmium tetroxide for 3 h. The fixed samples were consecutively treated with 2%osmium tetroxide for 2 h, 4%tannic acid for 8 h, and 2 % osmium tetroxide for 2 h. They were then dehydrated in a graded series of ethanols, immersed in t-butyl alcohol for 30 min freeze-dried in an Eiko ID-2 freeze-drying machine (Eiko Engineering, Mito, Japan), sputtercoated with platinum, and examined under a Jeol JSM-840A scanning electron microscope.
RESULTS The mean dimensions of the various parts of theguinea pig cochlear aqueduct were as follows: total length, 21.08 mm (range: 19.5-22.0 mm); maximum diameter of the opening to the subarachnoid space, 3.06 mm (range: 2.8-3.4 mm): a diameter of the narrowest segment, 58 pm (range: 40-80 pm): and the diameter of the opening to the perilymphatic space, 118 pm (range: 100-130 pm). The lumen of the cochlear aqueduct was widely contiguous with the subarachnoid space, contained CSF, and was traversed by delicate connective tissue trabeculae. The bony walls of the cochlear aqueduct were covered by both the dura mater and the arachnoid, which were continuous with the structures surrounding the brain (Fig. 1). The delicate connective tissue trabeculae were composed of blood vessels and fibroblasts, which showed a sheet-like appearance (Fig. 2). At the opening to the perilymphatic space of the scala tympani there was no actual barrier between the perilymphatic space and the Iuman ofthe cochlear aqueduct (Figs. 3 and 4). However, the fibroblasts of the connective tissue trabeculae were arranged more compactly and densely at this site than in the rest of the duct (Fig. 3). While these fibroblasts were arranged parallel to the long axis of the aqueduct in the main duct, they were perpendicular to the long axis at the opening to the perilymphatic space (Figs. 2 and 4). Scanning electron microscopy also detected no barrier at the opening of the cochlear aqueduct to the perilymphatic space (Figs. 5 and 6). There were many cribriform pores at the opening (Fig. 5) and the diameter of such pores was large enough for red blood cells to move freely through them (Fig. 6). Transmission electron microscopy showed the detailed structure of the connective tissue components of the trabeculae and the periotic tissues of the cochlear aqueduct. The connective tissue trabeculae were mainly composed of flattened and elongated fibroblasts and bundles of collagen fibrils. These fibroblasts were in contact with one another and formed a cellular network, while the bundles of collagen fibrils were closely associated with the fibroblasts and supported their network in the lumen of the cochlear aqueduct (Fig. 7). The periotic tissue were composed of multiple layers of both fibroblasts and densely arranged laminae of collagen fibrils (Fig. 8). These multilayered structures corresponded to the dura mater. Several inner layers of the periotic tissues were composed of thin flattened cell processes of spindleshaped cells and had no collagen fibrils between them (Fig. 8), and these layers corresponded to the arachnoid. Contacts between the fibroblasts belonging to the connective tissue trabeculae in the aqueduct lumen were clearly observed in thin sections. At some contact points, the intercellular space was 15-20 nm and was filled with rather electron-dense material, while the adjacent cytoplasm contained dense filamentous material. These structural features correspond to those of an intermediate junction (Fig. 9). Typical gap junctions were also frequently identified as septi-laminar membrane structures (Fig. 10). Many sites of punctate fusion of the plasma membrane were frequently observed between the fibroblasts, and these punctate
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Fig. 1. Low power view of the duct and its opening to the subarachnoid space. The lumen of the cochlear aqueduct is occupied by a sparse meshwork of cells. Note that the glossopharyngealnerve is located at the site of the opening to the subarachnoid space. x70. Fig. 2. The sparse meshwork of delicate connective tissue trabeculae is composed of both flattened fibroblasts and blood vessels. The fibroblasts are longitudinally arranged in the duct lumen. Note that several layers of the flattened cells cover the bony wall. ~ 2 8 0 . Fig. 3. Low power view of the opening to the perilymphatic space of scala tympani (So). The density of the fibroblasts seems to be more compact than in the main duct lumen. Arrows indicate the round window membrane. There is no bamer between the lumen of the cochlear aqueduct and the perilymphatic space (arrowheads). x 70. Fig. 4. No barrier can be seen at the opening to the perilymphatic space (arrowheads).Several layers of fibroblasts are arranged perpendicularly at the opening to the perilymphatic space. ~ 2 8 0 .
fusions usually may correspond to tight junctions with single or several strands (Fig. 10) ( 19-20). DISCUSSION Although the morphological features of the cochlear aqueduct have been investigated by many researchers, most of those studies used 20 to 25 pm serial sections of celloidin-mounted specimens (3, 1&15, 18). Since the mean diameter of the narrowest portion of the guinea pig cochlear aqueduct is 58 pm, 20 to 25 pm appears to be too thick to allow accurate investigation. In this study, we made semi-thin sections of epon-mounted specimens that were 0.2 to 0.5 pm thick for light microscopy. Thus, we could clearly demonstrate a sparse meshwork of fibroblasts and delicate connective tissue trabeculae in the lumen of the cochlear aqueduct.
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Figs. 5 and 6. Scanning electron microscopic view of the opening to the perilymphatic space of the scala tympani. There are many cribriform pores at the opening and these pores are large enough for red blood cells to move freely through them (arrowheuds). x220 and ~ 9 3 0 ,respectively.
These structural features were identical to those of the connective tissue components observed in semi-thin sections of spur resin-mounted subarachnoid tissue from the human brain (21 ). Transmission electron microscopy also showed that the ultrastructure of the delicate connective tissue trabeculae in the cochlear aqueduct was identical to that of the connective tissue of the subarachnoid space in the murine and human brains (20-23). Regarding the opening to the perilymphatic space of the scala tympani, the existence or otherwise of a barrier membrane has been an important problem. While some researchers reported the presence of a barrier membrane between the cochlear aqueduct and the perilymphatic space aAer studying serial sections of celloidin-mounted specimens (1 5 , 18). others could not find any such barrier structure (10, 14). In 1981, Nishimura et al. examined the tine structure of the guinea pig cochlear aqueduct using scanning electron microscopy and reported that there was no barrier membrane between the aqueduct and the perilymphatic space (2). In the present study, we reexamined the tine structure of the cribriform pores at the opening of the cochlear aqueduct to the perilymphatic space using scanning electron microscopy. We demonstrated that the sparse meshwork of fibroblasts and delicate connective tissue trabeculae was present even at the opening to the perilymphatic space, and that there was no specific bamer between the aqueduct and the perilymphatic space. However, we found that the fibroblasts of the connective tissue trabeculae in the cochlear aqueduct were more compact at
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TEM study of the guinea pig cochlear aqueduct 703
Fig. 7. Transmission electron microscopic view of the meshwork of fibroblasts. Many bundles of collagen fibrils are closely associated with the fibroblasts (urrows). Arrowheads indicate the inner few layers of the periotic tissue which correspond to the arachnoid. x 3 400.
Fig. 8. Transmission electron microscopic view of the periotic tissue lining the bony wall of the cochlear aqueduct. Note the multiple layers composed of flattened fibroblasts and densely arranged laminae of collagen fibrils. Arrowheads indicate the arachnoid. CF: collagen fibrils. x8 200.
the opening to the perilymphatic space than in the rest of the duct. Moreover, the arrangement of the fibroblasts at the opening to the perilymphatic space was perpendicular to the long axis of the aqueduct, whereas the fibroblasts in the main duct were parallel to the long axis. Thus, it is possible that in thick sections, such as serial sections of celloidin-mounted specimens, a pseudomembranous structure may sometimes be observed at the opening of the duct due to the density and the arrangement of the fibroblasts at this site. The periotic tissue lining the bony wall of the cochlear aqueduct has been morphologically examined using light and scanning electron microscopy (2, 3, 10, 11, 14, 16. 18). Although
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Figs. 9 and 10. High magnification view of cell-to-cell contacts in the meshwork of fibroblasts. Fig. 9. Arrows indicate that at some contacts the intercellular space is 15-20 nm and is filled with electrondense material, while the adjacent cytoplasm contains dense filamentous material. This type of contact is an intermediate junction. X36000. Fig. 10. Arrowheadr indicate typical gap junctions. Note that there are many punctate fusions of the plasma membrane, possibly tight junction, between the fibroblasts belonging to the connective tissue trabeculae (arrows). ~84000.
both the dura mater and the arachnoid have been observed to enter the cochlear aqueduct from the brain, the fine structure and the nature of the periotic tissue has not yet been determined using thin sections. In the present study, transmission electron microscopy of thin sections clearly demonstrated the fine morphological features of the periotic tissues. The total length of the periotic tissue, including the opening to the perilymphatic space, was a multilayered structure composed of both elongated cells and densely arranged laminae of collagen fibrils. These structural features of the periotic tissue are identical to those of the dura mater and the arachnoid (2&23). The fine structure of the periotic tissues of the guinea pig vestibular and cochlear perilymphatic spaces has been examined previously using decalcified specimens, and it has been proposed that the tissues lining these spaces were morphologically identical to the arachnoid and lacking a component corresponding to the dura mater (19,24). Thus, it may be concluded that the lumen of the cochlear aqueduct is the same as the subarachnoid space, while both the vestibular and cochlear perilymphatic spaces may correspond to an atypical subarachnoid space. The patency of the cochlear aqueduct has been examined in lower animals by tracer experiments (3, 11) and pressure-monitoring experiments (6-8), and patency has been generally accepted to be present. Our results morphologically support this concept. In addition, the
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TEA4 study of the guinea pig cochlear aqueduct 105
high density and the arrangement of the fibroblasts at the opening to the perilymphatic space suggest that the cochlear aqueduct may serve as a protective mechanism against sudden pressure changes in the CSF. Schuknecht & Reisser (9) have proposed that the perilymphatic ooze, which is encountered in stapes surgery for otosclerosis, is partly du to a widely patent cochlear aqueduct. Further experiments studying the ultrastructure of the human cochlear aqueduct are now underway at our laboratory.
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ACKNOWLEDGEMENTS We thank Ms Yuriko Arima for her helpful assistance and Mr T. Kanamaru for photographic help. This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education. Science and Culture (No.02857239).
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23. Krisch B, Leonhardt H. Oksche A. Compartments and perivascular arrangement of the meninges covering the cerebral cortex of the rat. Cell Tiss Res 1984 238: 459-74. 24. Franke K. Fine structure of the tissue lining the cochlear perilymphatic space against the bony labyrinthinecapsule. Arch Otolaryngol 1979; 222: 161-7. .Munuscript reeeir?pdOctober 18. 1990: accepted January 8. 1991
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Address for correspondence: Ryuzo Toriya, Department of Otolaryngology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812, Japan
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