Acta Oto-Laryngologica
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Sialomucin in Middle Ear Cholesteatoma Perimatrix Tomoyuki Nagai, Jun-Ichi Kawano & Tatsuo Suganuma To cite this article: Tomoyuki Nagai, Jun-Ichi Kawano & Tatsuo Suganuma (1992) Sialomucin in Middle Ear Cholesteatoma Perimatrix, Acta Oto-Laryngologica, 112:4, 686-690, DOI: 10.3109/00016489209137460 To link to this article: http://dx.doi.org/10.3109/00016489209137460
Published online: 08 Jul 2009.
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Date: 04 April 2016, At: 02:06
ACta O t O l a ~ g O (StWkh) l 1992; 112: 686-6%)
Sialomucin in Middle Ear Cholesteatoma Perimatrix TOMOYUKI NAGAI,’ JUN-ICHI KAWAN02 and TATSUO SUGANUMA’ From the Departments of ‘Othorhinolaryngology and 2Anatomy, M&maki Medical College, Miyazaki, 889-16 Japan
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Nagai T, Kawano J, Suganuma T. Sialomucin in middle ear cholesteatomaperimatrix. Acta Otolaryngol (Stockh) 1992; 112 686-690. Mucosubstance histochemistry of human middle ear cholesteatoma revealed that sialomucins are abundant and sulfomucins present in small amounts in the glandlike structures of the cholateatoma perimatrix. Based on the study, various glycoproteins were injected into the dermis of the external ear canal and infiltrated into the tympanic membranes of guinea pigs. Tympanic membranes were obtained 7 days later and light- and electron-microscopically studied. Injection of a sialomucin from bovine submaxillary gland resulted in marked proliferation of epidermis and degeneration of the lamina propria. Asialomucin prepared from sialomucin by hydrolysis produced mild thickening of the epidermis but the lamina propria was not degenerated. Fetuin and bovine serum albumin did not cause proliferation of the epidermis. Cholesterol granuloma formed in some of the specimens injected with sialomucin. The presence ofsialomucin in cholesteatomaperimatrix and these experimental studies using tympanic membranes of guinea pigs suggests that sialomucins participate in the proliferation of epidermis and degeneration of subepidermal connective tissue in human middle ear cholesteatoma. Key words: histochemistry, glycoproteinr, tympanic membrane, guinea pig, cholesterol granuloma.
INTRODUCTION The expansion of cholesteatoma obstructs the ventilation and drainage of the middle ear behind the cholesteatoma matrix. Confined mucous epithelium is present within a cholesteatoma perimatrix. In a previous paper (I), we demonstrated that the lymphatic vessels in the cholesteatoma perimatrix were obstructed by mucin-like substances, resulting in their degeneration. However, whether the degeneration of lymphatic vessels is caused by proteolytic activity, or toxic or osmotic action of mucin-like substances, is not clear. Middle ear effusions of children are more sticky than those of adults, and cholesteatoma of children is more aggressive than that of adults. The property of middle ear effusions and the aggressiveness of cholesteatoma in children may suggest that mucin in the cholesteatoma perimatrix causes proliferation of epidermis and degeneration of perimatrix. The present studies were performed to characterize human middle ear cholesteatoma by mucin-histochemistry and to confirm the role of mucin in cholesteatoma perimatrix by topical injection of various glycoproteins into the tympanic membranes of guinea pigs. MATERIAL AND METHODS
Human middle ear cholesteatoma tissues were obtained from 6 patients during ear surgery. These specimens were fixed in a mixture of 2% parafonnaldehyde and 2.5% glutaraldehyde solution, dehydrated in a graded series of ethyl alcohol and immersed in propylene oxide.
Then they were embedded in Epok 812. Thick sections cut at 1 pm were used for light microscopical study. Representative sections were stained with toluidine blue. For mucosubstance histochemistry, high iron diamine associated with alcian blue (pH 2.5) (HID-AB) was employed for the differentiation of sialomucins from sulfomucins (2). For the staining of the sections, removal of Epok resin (3) was performed before HID-AB staining.
Downloaded by [University of California Santa Barbara] at 02:06 04 April 2016
Acta Otolaryngol (Stockh) 112
Sialomuein in cholesteatoma 687
Sixty-two guinea pigs, weighing from 350 to 400 g were used for the following experimental studies. To examine the effect of mucin on the tympanic membranes, 1-2 mg per head of the following materials were injected: 1) Sialomucin from bovine submaxillary glands (Type I mucin, Sigma Chemical Co, St. Louis, MO, USA); 2) Sialomucin heated at 60°C for 1 h; 3) Asialomucin prepared from sialomucin by hydrolysis with 0.1 N H,SO, at 80°C for 30 min; 4) Fetuin from fetal calf serum (Sigma Chemical Co.), and 5 ) Bovine serum albumin (Sigma Chemical Co.). Under pentobarbikd anesthesia by intraperitoneal injection. 0.01-0.02 ml of physiological saline solution containing each material (lo%, W/V) was injected into the dermis of the external ear canal with a 27G needle. It was infiltrated into the tympanic membrane until a bulla was formed on the tympanic membrane. Seven days later the guinea pigs were killed under anesthesia by intraperitoneal injection of pentobarbital. The tympanic membranes were obtained and doubly fixed with 3% glutaraldehyde and 1% osmium tetroxide. They were dehydrated in a graded series of alcohol, immersed in propylene oxide and embedded in Epok 812. Thick sections were stained with toluidine blue. Thin sections were cut with a diamond knife attached to a Porter-Blum Type 1 microtome, doubly stained with uranyl acetate and lead nitrate, and examined with a JEOLZOOCX electron microscope. RESULTS Mucosubstance histochemistry of human cholesteatoma. Mucus glandlike structures were often found in the human cholesteatoma perimatrix (Fig. 1). The tubular structures were composed of two or three layers of continuous epithelial cells and lightly stained homogenous contents which sometimes leaked through chinks of the epithelial cells. The content of the glandlie structures was stained blue and faintly brown by high iron diamine associated with alcian blue, indicating that it contained a large quantity of sialomucins and a small quantity of sulfomucins (Fig. 2). Pathological changes of typmanic membranes induced by sialornucin. Topical injection of sialomucin increased the thickening of tympanic membranes. The hyperplastic epidermis was composed of 10 to 15 layers of keratinized squamous epithelial cells. Many layers of fibroblasts were present between the epidermis and the lamina propria (Fig. 3). The epidermis and subepidermal connective tissue were infiltrated by lymphocytes, neutrophils and macrophages. Injection of heat-treated sialomucin also resulted in the same findings (data not shown). In addition, asialomucin injection revealed a mild thickening of the tympanic membranes (data not shown). The epidermis was composed of 5 to 10 layers of keratinizing epithelium. The subepidermal connective tissue was composed of layers of fibroblasts and infiltrated by round cells. On the other hand, fetuin and bovine serum albumin produced only slight thickening of the tympanic membranes (Fig. 4). This epidermis had 2 to 3 layers of keratinizing epithelium. The subepidermal connective tissues were cellular in the case of injection of fetuin and edematous in the case of injection of bovine serum albumin. When sialomucin as well as heat-treated sialomucin were injected, collagen bundles of the lamina propria were loosened and disorganized (Fig. 5). Some fibroblasts within the lamina propria and subepidermal connective tissue contained rich endoplasmic reticulum and some contained large clear vacuoles, and yet others degenerated. Macrophages phagocytkl dense amorphous substances. Lymphatic vessels were not encountered in the specimens. Cholesterol granuloma was found in 2 out of 22 tympanic memebranes into which sialomucin was injected. Red blood cells were observed around the granuloma. Electron microscopical observation showed that the granuloma was composed of fibroblasts and macrophages which phagocytized cholesterol crystals (Fig. 6). Foreign body giant cells were not encountered.
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Fig. I . Glandlike structures in human cholesteatoma @matrix. The content is leaking out though a chink ( m o w ) . Toluidme blue staining. x 200. Fig. 2. HID-AB staining of human cholesteatoma perimatrix. Content of gland-like structures is stained. X
400.
Fig. 3. Sialomucin-injected tympanic membrane of guinea pig. Epidermis and subepidermal connective tissue show a marked thickening. Arrow indicates lamina propria. Toluidine blue staining. x 200. Fig. 4. Bovine serum albumin-injected tympanic membrane of guinea pig. Epidermis shows normal thickness but subepidermal connective tissue is dilated and contains faintly stained substances. Arrow indicates lamina propria. Toluidine blue staining. x200.
When asialomucin was injected 6ne collagen fibrils of the lamina propria were seen to be regularly arranged, and fibroblasts in the lamina propria and subepidermal connective tissue contained rich cytoplasmic organelles (Fig. 7). After injection of fetuin, macrophages infiltrated between the bundles of the lamina propria and phagocytized dense amorphous materials around the bundles (Fig. 8).
DISCUSSION The progressive growth of epidermis appears to be an important factor in the pathogenesis of cholesteatoma. O u r histochemical study of human cholesteatoma showed that sialomucin was dominantly present in glandlike structures and was leaking out into the cholesteatoma perimatrix. Our experimental study demonstrated that injection of sialomucin caused a marked proliferation of the epidermis of guinea pig tympanic membranes, compared to the injection of asialomucin. Therefore, it is suggested that sialomucin in the human cholesteatoma may cause the proliferation of epidermis. This is not a reaction to the injection of foreign bodies, because the proliferation of the epidermis did not occur when bovine serum albumin was injected. Topical injection of sialomucin into the typrnpanic membranes of the guinea pigs caused degeneration of the lamina propria and fibroblasts, whereas injection of asialomucin did not
Downloaded by [University of California Santa Barbara] at 02:06 04 April 2016
Downloaded by [University of California Santa Barbara] at 02:06 04 April 2016
690 T. Nagai et al.
Acta Otolaryngol (Stockh) 112
affect lamina propria at the ultrastructural level. These &dings suggest that sialomucin in the human cholesteatoma causes degeneration of the perimatrix. Degeneration of the lamina propria is not due to the protease unexpectedly contaminated in the sialomucin, because the sialomucin which was treated by heat raised the identical destruction of the lamina propna. Cholesterol crystals are found in the ear discharge from human middle ear cholesteatoma and cholesterol granuloma usually found behind the cholesteatoma. The origin of the cholesterol crystal is not well understmd. In our experiment, cholesterol granuloma was formed only after injection of sialomucin with association of hemorrhage within the tympanic membrane. Friedmann & Graham (4) has suggested from his electron microscopical study that cholesterol formed in the granuloma from blood, mucus or necrotic materials. Our experimental results support their hypothesis. The injection of sialomucin into the tympanic membranes caused pathological changes resembIing cholesteatoma: proliferation of the epidermis, degeneration of connective tissue and formation of cholesterol granuloma. Mucous middle ear effusions from pediatric patients contain abundant mucin type glycoproteins, whereas serous middle ear effusions from adult patients does not contain any mucin (5). Cholesteatoma of children is more aggressive than that of adults. Therefore, it is suggested that abundant secretion of mucin type glycoproteins from wnhed mucous epithelium in the cholesteatoma perimatrix may cause rapid expansion of cholesteatoma in children. REFERENCES 1. Nagai T, Kano K, Nagai M, Morimitsu T. The ultrastructure of lymphatic vessels in the human middle ear with cholesteatoma. Acta Otolaryngol (Stockh) 1989; 108: 442-7. 2. Spier SS. Diamine methods for differendating mucosubstances histochemidly. J Histochem Cytochem 1%5; 13: 211-34. 3. Maxwell MH.Two rapid and simple methods used for the removal of resins from 1.0 pm thick epoxy sections. J Microscopy 1977; 112: 253-5. 4. Fritdmann I, Graham MD.The ultrastructure of cholesterolgranuloma of the middle ear. J Laryngol Ot01 1979; 9 3 433-42. 5. Yabe R. Biochemical differences in middle ear effusions between pediatric and adult patients. 11. The protein composition (in Japanese). J Otolaryngol Jap 1991; 94: 525-33. Munuscript received Augvst 7, 1991; accepted October 29, 1991
Address for correspondence: TomoyuLi Nagai, Department of Otorhinolaryngology,Miyazaki Medical College, Kiyotake, Miyazaki, 889-16 Japan
Fig. 5. Electron microscopy of the lamina propria of heated sialomucin-injected tympanic membrane. Fibroblasts with rich cytoplasmic organelles (a). and with dialated endoplasmic reticulum (b) and degemrated ells (c) are observed between the unbound lamina propria. The macrophages contain dense substamzs (mow). x4,000.
Fig. 6. Electron microscopy of cholesterol granuloma. Cholesterol crystals (urrows)are phagocytkl by macrophap. F: Fibroblast. ~8,000. fig. Z Electron microscopy of the lamina propria of an asialomucin-injected tympanic membrane. The longitudinal section shows regular arrangement of collagen fibrils. The fibroblast contains abundant rough endoplasmic reticulum. x 5,000.
Fig. 8. Electron micr-py of the lamina propria of a fetuin-injected tympanic membrane. Macrophages and fibroblasts (F)are present between the bundles of collagen fibrils. The macrophages contain dense amorphous substances (arrow). x 6,000.
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Sialomucin in Middle Ear Cholesteatoma Perimatrix Tomoyuki Nagai, Jun-Ichi Kawano & Tatsuo Suganuma To cite this article: Tomoyuki Nagai, Jun-Ichi Kawano & Tatsuo Suganuma (1992) Sialomucin in Middle Ear Cholesteatoma Perimatrix, Acta Oto-Laryngologica, 112:4, 686-690, DOI: 10.3109/00016489209137460 To link to this article: http://dx.doi.org/10.3109/00016489209137460
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 2
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [University of California Santa Barbara]
Date: 04 April 2016, At: 02:06
ACta O t O l a ~ g O (StWkh) l 1992; 112: 686-6%)
Sialomucin in Middle Ear Cholesteatoma Perimatrix TOMOYUKI NAGAI,’ JUN-ICHI KAWAN02 and TATSUO SUGANUMA’ From the Departments of ‘Othorhinolaryngology and 2Anatomy, M&maki Medical College, Miyazaki, 889-16 Japan
Downloaded by [University of California Santa Barbara] at 02:06 04 April 2016
Nagai T, Kawano J, Suganuma T. Sialomucin in middle ear cholesteatomaperimatrix. Acta Otolaryngol (Stockh) 1992; 112 686-690. Mucosubstance histochemistry of human middle ear cholesteatoma revealed that sialomucins are abundant and sulfomucins present in small amounts in the glandlike structures of the cholateatoma perimatrix. Based on the study, various glycoproteins were injected into the dermis of the external ear canal and infiltrated into the tympanic membranes of guinea pigs. Tympanic membranes were obtained 7 days later and light- and electron-microscopically studied. Injection of a sialomucin from bovine submaxillary gland resulted in marked proliferation of epidermis and degeneration of the lamina propria. Asialomucin prepared from sialomucin by hydrolysis produced mild thickening of the epidermis but the lamina propria was not degenerated. Fetuin and bovine serum albumin did not cause proliferation of the epidermis. Cholesterol granuloma formed in some of the specimens injected with sialomucin. The presence ofsialomucin in cholesteatomaperimatrix and these experimental studies using tympanic membranes of guinea pigs suggests that sialomucins participate in the proliferation of epidermis and degeneration of subepidermal connective tissue in human middle ear cholesteatoma. Key words: histochemistry, glycoproteinr, tympanic membrane, guinea pig, cholesterol granuloma.
INTRODUCTION The expansion of cholesteatoma obstructs the ventilation and drainage of the middle ear behind the cholesteatoma matrix. Confined mucous epithelium is present within a cholesteatoma perimatrix. In a previous paper (I), we demonstrated that the lymphatic vessels in the cholesteatoma perimatrix were obstructed by mucin-like substances, resulting in their degeneration. However, whether the degeneration of lymphatic vessels is caused by proteolytic activity, or toxic or osmotic action of mucin-like substances, is not clear. Middle ear effusions of children are more sticky than those of adults, and cholesteatoma of children is more aggressive than that of adults. The property of middle ear effusions and the aggressiveness of cholesteatoma in children may suggest that mucin in the cholesteatoma perimatrix causes proliferation of epidermis and degeneration of perimatrix. The present studies were performed to characterize human middle ear cholesteatoma by mucin-histochemistry and to confirm the role of mucin in cholesteatoma perimatrix by topical injection of various glycoproteins into the tympanic membranes of guinea pigs. MATERIAL AND METHODS
Human middle ear cholesteatoma tissues were obtained from 6 patients during ear surgery. These specimens were fixed in a mixture of 2% parafonnaldehyde and 2.5% glutaraldehyde solution, dehydrated in a graded series of ethyl alcohol and immersed in propylene oxide.
Then they were embedded in Epok 812. Thick sections cut at 1 pm were used for light microscopical study. Representative sections were stained with toluidine blue. For mucosubstance histochemistry, high iron diamine associated with alcian blue (pH 2.5) (HID-AB) was employed for the differentiation of sialomucins from sulfomucins (2). For the staining of the sections, removal of Epok resin (3) was performed before HID-AB staining.
Downloaded by [University of California Santa Barbara] at 02:06 04 April 2016
Acta Otolaryngol (Stockh) 112
Sialomuein in cholesteatoma 687
Sixty-two guinea pigs, weighing from 350 to 400 g were used for the following experimental studies. To examine the effect of mucin on the tympanic membranes, 1-2 mg per head of the following materials were injected: 1) Sialomucin from bovine submaxillary glands (Type I mucin, Sigma Chemical Co, St. Louis, MO, USA); 2) Sialomucin heated at 60°C for 1 h; 3) Asialomucin prepared from sialomucin by hydrolysis with 0.1 N H,SO, at 80°C for 30 min; 4) Fetuin from fetal calf serum (Sigma Chemical Co.), and 5 ) Bovine serum albumin (Sigma Chemical Co.). Under pentobarbikd anesthesia by intraperitoneal injection. 0.01-0.02 ml of physiological saline solution containing each material (lo%, W/V) was injected into the dermis of the external ear canal with a 27G needle. It was infiltrated into the tympanic membrane until a bulla was formed on the tympanic membrane. Seven days later the guinea pigs were killed under anesthesia by intraperitoneal injection of pentobarbital. The tympanic membranes were obtained and doubly fixed with 3% glutaraldehyde and 1% osmium tetroxide. They were dehydrated in a graded series of alcohol, immersed in propylene oxide and embedded in Epok 812. Thick sections were stained with toluidine blue. Thin sections were cut with a diamond knife attached to a Porter-Blum Type 1 microtome, doubly stained with uranyl acetate and lead nitrate, and examined with a JEOLZOOCX electron microscope. RESULTS Mucosubstance histochemistry of human cholesteatoma. Mucus glandlike structures were often found in the human cholesteatoma perimatrix (Fig. 1). The tubular structures were composed of two or three layers of continuous epithelial cells and lightly stained homogenous contents which sometimes leaked through chinks of the epithelial cells. The content of the glandlie structures was stained blue and faintly brown by high iron diamine associated with alcian blue, indicating that it contained a large quantity of sialomucins and a small quantity of sulfomucins (Fig. 2). Pathological changes of typmanic membranes induced by sialornucin. Topical injection of sialomucin increased the thickening of tympanic membranes. The hyperplastic epidermis was composed of 10 to 15 layers of keratinized squamous epithelial cells. Many layers of fibroblasts were present between the epidermis and the lamina propria (Fig. 3). The epidermis and subepidermal connective tissue were infiltrated by lymphocytes, neutrophils and macrophages. Injection of heat-treated sialomucin also resulted in the same findings (data not shown). In addition, asialomucin injection revealed a mild thickening of the tympanic membranes (data not shown). The epidermis was composed of 5 to 10 layers of keratinizing epithelium. The subepidermal connective tissue was composed of layers of fibroblasts and infiltrated by round cells. On the other hand, fetuin and bovine serum albumin produced only slight thickening of the tympanic membranes (Fig. 4). This epidermis had 2 to 3 layers of keratinizing epithelium. The subepidermal connective tissues were cellular in the case of injection of fetuin and edematous in the case of injection of bovine serum albumin. When sialomucin as well as heat-treated sialomucin were injected, collagen bundles of the lamina propria were loosened and disorganized (Fig. 5). Some fibroblasts within the lamina propria and subepidermal connective tissue contained rich endoplasmic reticulum and some contained large clear vacuoles, and yet others degenerated. Macrophages phagocytkl dense amorphous substances. Lymphatic vessels were not encountered in the specimens. Cholesterol granuloma was found in 2 out of 22 tympanic memebranes into which sialomucin was injected. Red blood cells were observed around the granuloma. Electron microscopical observation showed that the granuloma was composed of fibroblasts and macrophages which phagocytized cholesterol crystals (Fig. 6). Foreign body giant cells were not encountered.
Downloaded by [University of California Santa Barbara] at 02:06 04 April 2016
688
T.Nugui
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Acta Otolaryngol (Stockh) 112
Fig. I . Glandlike structures in human cholesteatoma @matrix. The content is leaking out though a chink ( m o w ) . Toluidme blue staining. x 200. Fig. 2. HID-AB staining of human cholesteatoma perimatrix. Content of gland-like structures is stained. X
400.
Fig. 3. Sialomucin-injected tympanic membrane of guinea pig. Epidermis and subepidermal connective tissue show a marked thickening. Arrow indicates lamina propria. Toluidine blue staining. x 200. Fig. 4. Bovine serum albumin-injected tympanic membrane of guinea pig. Epidermis shows normal thickness but subepidermal connective tissue is dilated and contains faintly stained substances. Arrow indicates lamina propria. Toluidine blue staining. x200.
When asialomucin was injected 6ne collagen fibrils of the lamina propria were seen to be regularly arranged, and fibroblasts in the lamina propria and subepidermal connective tissue contained rich cytoplasmic organelles (Fig. 7). After injection of fetuin, macrophages infiltrated between the bundles of the lamina propria and phagocytized dense amorphous materials around the bundles (Fig. 8).
DISCUSSION The progressive growth of epidermis appears to be an important factor in the pathogenesis of cholesteatoma. O u r histochemical study of human cholesteatoma showed that sialomucin was dominantly present in glandlike structures and was leaking out into the cholesteatoma perimatrix. Our experimental study demonstrated that injection of sialomucin caused a marked proliferation of the epidermis of guinea pig tympanic membranes, compared to the injection of asialomucin. Therefore, it is suggested that sialomucin in the human cholesteatoma may cause the proliferation of epidermis. This is not a reaction to the injection of foreign bodies, because the proliferation of the epidermis did not occur when bovine serum albumin was injected. Topical injection of sialomucin into the typrnpanic membranes of the guinea pigs caused degeneration of the lamina propria and fibroblasts, whereas injection of asialomucin did not
Downloaded by [University of California Santa Barbara] at 02:06 04 April 2016
Downloaded by [University of California Santa Barbara] at 02:06 04 April 2016
690 T. Nagai et al.
Acta Otolaryngol (Stockh) 112
affect lamina propria at the ultrastructural level. These &dings suggest that sialomucin in the human cholesteatoma causes degeneration of the perimatrix. Degeneration of the lamina propria is not due to the protease unexpectedly contaminated in the sialomucin, because the sialomucin which was treated by heat raised the identical destruction of the lamina propna. Cholesterol crystals are found in the ear discharge from human middle ear cholesteatoma and cholesterol granuloma usually found behind the cholesteatoma. The origin of the cholesterol crystal is not well understmd. In our experiment, cholesterol granuloma was formed only after injection of sialomucin with association of hemorrhage within the tympanic membrane. Friedmann & Graham (4) has suggested from his electron microscopical study that cholesterol formed in the granuloma from blood, mucus or necrotic materials. Our experimental results support their hypothesis. The injection of sialomucin into the tympanic membranes caused pathological changes resembIing cholesteatoma: proliferation of the epidermis, degeneration of connective tissue and formation of cholesterol granuloma. Mucous middle ear effusions from pediatric patients contain abundant mucin type glycoproteins, whereas serous middle ear effusions from adult patients does not contain any mucin (5). Cholesteatoma of children is more aggressive than that of adults. Therefore, it is suggested that abundant secretion of mucin type glycoproteins from wnhed mucous epithelium in the cholesteatoma perimatrix may cause rapid expansion of cholesteatoma in children. REFERENCES 1. Nagai T, Kano K, Nagai M, Morimitsu T. The ultrastructure of lymphatic vessels in the human middle ear with cholesteatoma. Acta Otolaryngol (Stockh) 1989; 108: 442-7. 2. Spier SS. Diamine methods for differendating mucosubstances histochemidly. J Histochem Cytochem 1%5; 13: 211-34. 3. Maxwell MH.Two rapid and simple methods used for the removal of resins from 1.0 pm thick epoxy sections. J Microscopy 1977; 112: 253-5. 4. Fritdmann I, Graham MD.The ultrastructure of cholesterolgranuloma of the middle ear. J Laryngol Ot01 1979; 9 3 433-42. 5. Yabe R. Biochemical differences in middle ear effusions between pediatric and adult patients. 11. The protein composition (in Japanese). J Otolaryngol Jap 1991; 94: 525-33. Munuscript received Augvst 7, 1991; accepted October 29, 1991
Address for correspondence: TomoyuLi Nagai, Department of Otorhinolaryngology,Miyazaki Medical College, Kiyotake, Miyazaki, 889-16 Japan
Fig. 5. Electron microscopy of the lamina propria of heated sialomucin-injected tympanic membrane. Fibroblasts with rich cytoplasmic organelles (a). and with dialated endoplasmic reticulum (b) and degemrated ells (c) are observed between the unbound lamina propria. The macrophages contain dense substamzs (mow). x4,000.
Fig. 6. Electron microscopy of cholesterol granuloma. Cholesterol crystals (urrows)are phagocytkl by macrophap. F: Fibroblast. ~8,000. fig. Z Electron microscopy of the lamina propria of an asialomucin-injected tympanic membrane. The longitudinal section shows regular arrangement of collagen fibrils. The fibroblast contains abundant rough endoplasmic reticulum. x 5,000.
Fig. 8. Electron micr-py of the lamina propria of a fetuin-injected tympanic membrane. Macrophages and fibroblasts (F)are present between the bundles of collagen fibrils. The macrophages contain dense amorphous substances (arrow). x 6,000.
