Ann Otal 84: 1975
MUCOSAL HISTOCHEMISTRY IN SECRETORY OTITIS TAUNO PALVA,
M.D.
HELSINKI, FINLAND
ANTTI PALVA,
M.D.
OULU, FINLAND
SUMMARY - Mucosal biopsies were taken from 20 ears with secretory otitis media (glue ear) and histochemical stainings were made for comparison with data obtained from biochemical analysis of the fluids. Acid phosphatase, lactate dehydrogenase (LD), and malate dehydrogenase (MD), the activity of which in the ear fluids was 20 to 30 times higher than in serum, were found to appear as strong precipitates in the middle ear epithelium, particularly in the top layer. Alkaline phosphatase activity was only exceptionally seen in the epithelium but appeared in the capillaries and histiocytes. Nonspecific esterase appeared irregularly in the epithelium and regularly in histioeytes. The latter two had lower activities in ear fluids than in serum. Epithelial secretory cells and subepithelial glands and cysts showed strong alcian blue (AB )-positive staining. Positive material appeared also in the cytoplasm of the epithelial cells and in the intercellular substance. Distinct PAS-positive staining appeared in the columnar epithelium and particularly in the free mucus on top of the epithelium but was less pronounced in the glandular structures and absent from the cysts.
Histochemical studies of the middle ear mucosa in secretory otitis media have shown an increase of secretory cells and glandsv" as compared with normal middle ear epithelium. These secretory elements produce varying amounts of PAS and alcian blue (AB) positive mucus in the middle ear cavity. An increased metabolic activity of the mucosa has also been demonstrated in the form of high acid phosphatase activity of the epithelium."
water," To obtain more information, we have continued our histochemical analyses of middle ear mucosal biopsies" in cases of secretory otitis media. If the epithelium actively secretes some enzymes into the ear fluid, then there must also be activity in the epithelium itself. METHODS AND MATERIALS
In the ear fluid itself a distinct activity increase of many enzymes has been found, as compared to the serum levels. This applies to lactate (LD) and malate dehydrogenases (MD) and to acid phosphatasesv" whereas alkaline phosphatase activity has been similar to that in serum and nonspecific esterases have shown higher activity in serum than in the ear fluid. 6 The increase of enzyme activities in ear fluid were 20 to 30 times higher than in serum'' and this difference is too great to be accounted for merely by reabsorption of
Using small microforceps, biopsies from middle ear mucosa covering the promontory were taken from 20 ears in connection with myringotomy and insertion of a ventilation tube. The small mucosal pieces were immersed in Lipshaw M-I embedding matrix on a cork slice, frozen in liquid nitrogen, and stored at _70 The sections were cut at -23°C with a cold microtome to 7 /L. Staining for acid and alkaline phosphatases, for LD, MD and for nonspecific esterases was done as described earlier.s Staining for PAS and AB was made according to the directions given in Pearse.P 0
•
RESULTS
Hematoxylin and Eosin Staining. This staining showed that the biopsy pieces contained secretory epithelial cells, su-
---From the Department of Otolaryngology, University of Oulu, Finland. 112
Downloaded from aor.sagepub.com at DALHOUSIE UNIV on June 5, 2016
SECRETORY OTITIS
Fig. 1. Acid phosphatase staining: The four-cell layer columnar epithelium shows some positive staining throughout; but the activity is strongest in the superficial cells, and a black precipitate is seen to cover the surface of the cells.
perflcial and deep secretory glands and a few comparatively large mucus cysts with a thin epithelium of one to two layers. Ciliated columnar cells appeared in these biopsies only exceptionally and the epithelium was generally high columnar or low cuboidal consisting of three or four layers. Squamous epithelium was never seen except as a contaminant from the edges of the myringotomy incision brought forward by the biopsy forceps. In addition to the glands, histiocytes and lymphocytes appeared in the stroma and an increase of young fibrocytes was noticeable.
Staining for Acid Phosphatase. Distinct acid phosphatase activity was found in most areas throughout the higher columnar epithelium, the greatest activity being concentrated to the apical parts of the cells in the superficial layer (Fig. 1). Secretion on top of the cells as well as the mucus adjoining the biopsy pieces showed strong staining. However, certain areas of low cuboidal epithelium were free of activity, and in other areas only the superficial epithelial cells showed enzyme precipitate. Secretory glands and cysts
113
Fig. 2. Alkaline phosphatase staining: The capillaries and histiocytic cells show strong staining while the columnar epithelial cells are devoid of activity. Irregular patterns of positive precipitate are seen in the areas of young fibrocytes. Magnification 150X.
were devoid of activity. Histiocytic cells in the stroma showed positive acid phosphatase staining.
Staining for Alkaline Phosphatase. The biopsy pieces were, as a rule, well vascularized and alkaline phosphatase activity therefore appeared both in capillary cross and longitudinal sections. Columnar epithelial cells were almost devoid of activity as were also the glandular structures and cysts. Histiocytes showed active staining. In the free mucus adjacent to the biopsy pieces, a faint enzyme activity could be discerned. Some areas of young, active fibrocytes were surrounded by alkaline phosphatase positive precipitate showing irregular patterns (Fig. 2). Staining for Lactate Dehydrogenase. All biopsy pieces showed very marked activity in the form of a granular precipitate. Staining was strongest in the columnar epithelium cells, particularly in the top layer, but also the stroma
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PALVA-PALVA
Fig. 3. Lactate dehydrogenase staining: An intense activity is seen in the whole epithelial layer and a moderate activity is seen also all over the stroma. Magnification 250X.
Fig. 5. An intense AB positive staining of two active secretory stromal glands. Magnification 250X.
showed marked over-all activity (Fig. 3). The cystic cavities showed no staining and the secretory glands no remarkable activity. Distinct granular staining was seen also in the mucus surrounding the biopsy pieces.
Staining for Malate Dehydrogenase. All that is stated above of LD applies also to MD except that the intensity of activity for the latter seemed to be slightly less than for LD. Staining for Nonspecific Esterase. Staining for nonspecific esterase was distinct in some limited areas of the columnar epithelium, appearing as patchy dark precipitates (Fig. 4). However, large areas of epithelium were totally free of activity. No activity appeared either in the glands or in the cysts. Stromal enzyme staining was absent except in the histiocytes, which stained also in the adjoining areas of mucus. Fig. 4. Nonspecific esterase staining: Secretory epithelial cells show distinct activity in the upper part of the specimen but epithelium at bottom shows no activity. Histiocytic cells in the stroma stain clearly. Magnification 250X.
Alcian Blue Staining. A beautiful intense AB-positive staining was seen in the secretion filling the lumen and in the ducts of the secretory glands and the cysts (Fig. 5). In the cytoplasm of the secretory cells in the epithelium, an
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SECRETORY OTITIS
115
Fig. 6. Strong epithelial secretory cell staining by AB. Magnification 6OOX.
intense staining was noted (Fig. 6). AB-positive staining was also found in the intercellular space throughout the stroma in varying extent. The surface of the columnar epithelium was often covered with a distinct blue mucus lining.
Periodic Acid Schiff Staining. The strongest PAS-positive staining appeared in the mucus adjoining the biopsy pieces. In the superficial layer of the columnar epithelium there were areas of patchy red staining of the cytoplasm but many areas were PASnegative (Fig. 7). Strands of distinct PAS-positive material occurred in the narrow lumina of the secretory glands whereas the cysts stained negative. DISCUSSION
This histochemical study of small biopsy specimens from promontory mucosa in glue ears has confirmed the data presented earlier on the basis of the fluid analyses.s-" Thus acid phosphatase activity, which appeared distinctly in the columnar epithelium and particularly in its top layer, was also seen in the free mucus. This is undoubtedly a result of epithelial secretory activity. A similar relationship was found in the case of LD and MD, both of which showed intense activity in the whole biopsy specimen but particularly in the epithelium. Two other enzymes, viz.,
Fig. 7. PAS-staining showing strong activity of the epithelial secretory cells. Magnification 600X.
alkaline phosphatase and nonspecific esterase," which in the quantitative analyses revealed an activity equal to or lower than serum, were only exceptionally demonstrable in the epithelium. Alkaline phosphatase activity appeared in the capillaries and histiocytes. Nonspecific esterase activity in the specimens was uneven, some areas showing distinct epithelial activity and some none. The hulk of the secreted mucus apparently consists of mucopolysaccharides since, in the retention cysts and in the active deep and superficial glands, all mucus was strongly AB-positive. Acid mucopolysaccharides also appeared in the epithelial and secretory cell cytoplasm and in the intercellular substance. PAS-positive mucus, indicating the presence of neutral mucopolysaccharides, appeared in the epithelium and in the free mucus adjoining the biopsy pieces; but its share in the products of the secretory glands was clearly less than that of acid mucopolysaccharides. In general, the PAS-AB positivity in the glands of the mucosa in cases of secretory otitis is an established finding,2,lO.12 and the middle
Downloaded from aor.sagepub.com at DALHOUSIE UNIV on June 5, 2016
116
PALVA-PALVA
ear mucosa seems to play an active part in the continuous production of mucus in the glue ear. The IgA type mucoantibodies'P-t! apparently play an important role in the defense mechanism of the middle ear. Although some studies report on the basis of elaborate tests that the effusion develops through capillary leakage and is of serum origin.l! it is evident that in glue ears this is not the case. The mucosa itself produces much of the material secreted into the middle ear
space and is unable to remove it via the Eustachian tube. Whether this is due to a lack of adequate ciliary mechanism cannot be answered at this stage. Our small biopsy specimens showed a conspicuous absence of ciliated epithelium, but this is not uncommon in the area where the biopsies were taken. It is to be hoped that some temporal bones can be obtained later to make possible a comparison with normal data on the amount of ciliated cell tracks in the middle ear mucosa.
DEPARTMENT OF OTOLARYNGOLOGY, UNIVERSITY OF OULU,
Om.c, FINLAND.
REFERENCES I. Friedmann I: The pathology of secretory otitis media. Proc R Soc Med 56:695-699, 1963 2. Sade J: Pathology and pathogenesis of serous otitis media. Arch Otolaryngol 84:297305, 1966 3. Lim DJ, Viall J, Birck H, et al: Symposium on prophylaxis and treatment of middle ear effusions. Laryngoscope 82: 1625-1642, 1972 4. Tos M, Bak-Pedersen K: Density of mucous glands in a biopsy material of chronic secretory otitis media. Acta Otolaryngol (Stockh) 75:55-60, 1973 5. juhn SK, Huff JS, Paparella MM: Biochemical analyses of middle ear effusions. Ann Otol Rhinol Laryngol 80:347-353, 1971 6. Paiva T, Raunio V, Nousianen R: Secretory otitis media: protein and enzyme analyses. Ann Otol Rhinol Laryngol 83:35-43 (Suppl II),1974 7. Carlson LA, Lokk T: Protein studies of transudates of the middle ear. Scand J Clin Lab Invest 7:43-48, 1955
8. Paiva T, Paiva A, Dammert K: Middle ear mucosa and chronic ear disease. Arch Otolaryngol 91:50-56, 1970 9. Pearse, AGE: Histochemistry. London, J and A Churchill Ltd, 1960 10. Gundersen T, Gluck E: The middle ear mucosa in serous otitis media. Arch OtoIaryngol 96:40-44, 1972 II. Tos M, Bak-Pedersen K: New aspects in the pathogenesis of chronic secretory otitis media. Acta Otolaryngol (Stockh) 75:269270, 1973 12. Bernstein JM, Hayes ER, Ishikawa T, et al: Secretory otitis media: a histopathologic and immunochemical report. Trans Am Acad Ophthalmol Otolaryngol 76:1305-1318, 1972 13. Mogi G, Hon]o S, Maeda S, et al: Secretory immunoglobulin A (SIgA) in middle ear effusions. Ann Otol Rhinol Laryngol 82:302-310, 1973 14. Tender 0, Gundersen T: Nature of the fluid in serous otitis media. Arch Otolaryngol 93:473-478, 1971
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MUCOSAL HISTOCHEMISTRY IN SECRETORY OTITIS TAUNO PALVA,
M.D.
HELSINKI, FINLAND
ANTTI PALVA,
M.D.
OULU, FINLAND
SUMMARY - Mucosal biopsies were taken from 20 ears with secretory otitis media (glue ear) and histochemical stainings were made for comparison with data obtained from biochemical analysis of the fluids. Acid phosphatase, lactate dehydrogenase (LD), and malate dehydrogenase (MD), the activity of which in the ear fluids was 20 to 30 times higher than in serum, were found to appear as strong precipitates in the middle ear epithelium, particularly in the top layer. Alkaline phosphatase activity was only exceptionally seen in the epithelium but appeared in the capillaries and histiocytes. Nonspecific esterase appeared irregularly in the epithelium and regularly in histioeytes. The latter two had lower activities in ear fluids than in serum. Epithelial secretory cells and subepithelial glands and cysts showed strong alcian blue (AB )-positive staining. Positive material appeared also in the cytoplasm of the epithelial cells and in the intercellular substance. Distinct PAS-positive staining appeared in the columnar epithelium and particularly in the free mucus on top of the epithelium but was less pronounced in the glandular structures and absent from the cysts.
Histochemical studies of the middle ear mucosa in secretory otitis media have shown an increase of secretory cells and glandsv" as compared with normal middle ear epithelium. These secretory elements produce varying amounts of PAS and alcian blue (AB) positive mucus in the middle ear cavity. An increased metabolic activity of the mucosa has also been demonstrated in the form of high acid phosphatase activity of the epithelium."
water," To obtain more information, we have continued our histochemical analyses of middle ear mucosal biopsies" in cases of secretory otitis media. If the epithelium actively secretes some enzymes into the ear fluid, then there must also be activity in the epithelium itself. METHODS AND MATERIALS
In the ear fluid itself a distinct activity increase of many enzymes has been found, as compared to the serum levels. This applies to lactate (LD) and malate dehydrogenases (MD) and to acid phosphatasesv" whereas alkaline phosphatase activity has been similar to that in serum and nonspecific esterases have shown higher activity in serum than in the ear fluid. 6 The increase of enzyme activities in ear fluid were 20 to 30 times higher than in serum'' and this difference is too great to be accounted for merely by reabsorption of
Using small microforceps, biopsies from middle ear mucosa covering the promontory were taken from 20 ears in connection with myringotomy and insertion of a ventilation tube. The small mucosal pieces were immersed in Lipshaw M-I embedding matrix on a cork slice, frozen in liquid nitrogen, and stored at _70 The sections were cut at -23°C with a cold microtome to 7 /L. Staining for acid and alkaline phosphatases, for LD, MD and for nonspecific esterases was done as described earlier.s Staining for PAS and AB was made according to the directions given in Pearse.P 0
•
RESULTS
Hematoxylin and Eosin Staining. This staining showed that the biopsy pieces contained secretory epithelial cells, su-
---From the Department of Otolaryngology, University of Oulu, Finland. 112
Downloaded from aor.sagepub.com at DALHOUSIE UNIV on June 5, 2016
SECRETORY OTITIS
Fig. 1. Acid phosphatase staining: The four-cell layer columnar epithelium shows some positive staining throughout; but the activity is strongest in the superficial cells, and a black precipitate is seen to cover the surface of the cells.
perflcial and deep secretory glands and a few comparatively large mucus cysts with a thin epithelium of one to two layers. Ciliated columnar cells appeared in these biopsies only exceptionally and the epithelium was generally high columnar or low cuboidal consisting of three or four layers. Squamous epithelium was never seen except as a contaminant from the edges of the myringotomy incision brought forward by the biopsy forceps. In addition to the glands, histiocytes and lymphocytes appeared in the stroma and an increase of young fibrocytes was noticeable.
Staining for Acid Phosphatase. Distinct acid phosphatase activity was found in most areas throughout the higher columnar epithelium, the greatest activity being concentrated to the apical parts of the cells in the superficial layer (Fig. 1). Secretion on top of the cells as well as the mucus adjoining the biopsy pieces showed strong staining. However, certain areas of low cuboidal epithelium were free of activity, and in other areas only the superficial epithelial cells showed enzyme precipitate. Secretory glands and cysts
113
Fig. 2. Alkaline phosphatase staining: The capillaries and histiocytic cells show strong staining while the columnar epithelial cells are devoid of activity. Irregular patterns of positive precipitate are seen in the areas of young fibrocytes. Magnification 150X.
were devoid of activity. Histiocytic cells in the stroma showed positive acid phosphatase staining.
Staining for Alkaline Phosphatase. The biopsy pieces were, as a rule, well vascularized and alkaline phosphatase activity therefore appeared both in capillary cross and longitudinal sections. Columnar epithelial cells were almost devoid of activity as were also the glandular structures and cysts. Histiocytes showed active staining. In the free mucus adjacent to the biopsy pieces, a faint enzyme activity could be discerned. Some areas of young, active fibrocytes were surrounded by alkaline phosphatase positive precipitate showing irregular patterns (Fig. 2). Staining for Lactate Dehydrogenase. All biopsy pieces showed very marked activity in the form of a granular precipitate. Staining was strongest in the columnar epithelium cells, particularly in the top layer, but also the stroma
Downloaded from aor.sagepub.com at DALHOUSIE UNIV on June 5, 2016
114
PALVA-PALVA
Fig. 3. Lactate dehydrogenase staining: An intense activity is seen in the whole epithelial layer and a moderate activity is seen also all over the stroma. Magnification 250X.
Fig. 5. An intense AB positive staining of two active secretory stromal glands. Magnification 250X.
showed marked over-all activity (Fig. 3). The cystic cavities showed no staining and the secretory glands no remarkable activity. Distinct granular staining was seen also in the mucus surrounding the biopsy pieces.
Staining for Malate Dehydrogenase. All that is stated above of LD applies also to MD except that the intensity of activity for the latter seemed to be slightly less than for LD. Staining for Nonspecific Esterase. Staining for nonspecific esterase was distinct in some limited areas of the columnar epithelium, appearing as patchy dark precipitates (Fig. 4). However, large areas of epithelium were totally free of activity. No activity appeared either in the glands or in the cysts. Stromal enzyme staining was absent except in the histiocytes, which stained also in the adjoining areas of mucus. Fig. 4. Nonspecific esterase staining: Secretory epithelial cells show distinct activity in the upper part of the specimen but epithelium at bottom shows no activity. Histiocytic cells in the stroma stain clearly. Magnification 250X.
Alcian Blue Staining. A beautiful intense AB-positive staining was seen in the secretion filling the lumen and in the ducts of the secretory glands and the cysts (Fig. 5). In the cytoplasm of the secretory cells in the epithelium, an
Downloaded from aor.sagepub.com at DALHOUSIE UNIV on June 5, 2016
SECRETORY OTITIS
115
Fig. 6. Strong epithelial secretory cell staining by AB. Magnification 6OOX.
intense staining was noted (Fig. 6). AB-positive staining was also found in the intercellular space throughout the stroma in varying extent. The surface of the columnar epithelium was often covered with a distinct blue mucus lining.
Periodic Acid Schiff Staining. The strongest PAS-positive staining appeared in the mucus adjoining the biopsy pieces. In the superficial layer of the columnar epithelium there were areas of patchy red staining of the cytoplasm but many areas were PASnegative (Fig. 7). Strands of distinct PAS-positive material occurred in the narrow lumina of the secretory glands whereas the cysts stained negative. DISCUSSION
This histochemical study of small biopsy specimens from promontory mucosa in glue ears has confirmed the data presented earlier on the basis of the fluid analyses.s-" Thus acid phosphatase activity, which appeared distinctly in the columnar epithelium and particularly in its top layer, was also seen in the free mucus. This is undoubtedly a result of epithelial secretory activity. A similar relationship was found in the case of LD and MD, both of which showed intense activity in the whole biopsy specimen but particularly in the epithelium. Two other enzymes, viz.,
Fig. 7. PAS-staining showing strong activity of the epithelial secretory cells. Magnification 600X.
alkaline phosphatase and nonspecific esterase," which in the quantitative analyses revealed an activity equal to or lower than serum, were only exceptionally demonstrable in the epithelium. Alkaline phosphatase activity appeared in the capillaries and histiocytes. Nonspecific esterase activity in the specimens was uneven, some areas showing distinct epithelial activity and some none. The hulk of the secreted mucus apparently consists of mucopolysaccharides since, in the retention cysts and in the active deep and superficial glands, all mucus was strongly AB-positive. Acid mucopolysaccharides also appeared in the epithelial and secretory cell cytoplasm and in the intercellular substance. PAS-positive mucus, indicating the presence of neutral mucopolysaccharides, appeared in the epithelium and in the free mucus adjoining the biopsy pieces; but its share in the products of the secretory glands was clearly less than that of acid mucopolysaccharides. In general, the PAS-AB positivity in the glands of the mucosa in cases of secretory otitis is an established finding,2,lO.12 and the middle
Downloaded from aor.sagepub.com at DALHOUSIE UNIV on June 5, 2016
116
PALVA-PALVA
ear mucosa seems to play an active part in the continuous production of mucus in the glue ear. The IgA type mucoantibodies'P-t! apparently play an important role in the defense mechanism of the middle ear. Although some studies report on the basis of elaborate tests that the effusion develops through capillary leakage and is of serum origin.l! it is evident that in glue ears this is not the case. The mucosa itself produces much of the material secreted into the middle ear
space and is unable to remove it via the Eustachian tube. Whether this is due to a lack of adequate ciliary mechanism cannot be answered at this stage. Our small biopsy specimens showed a conspicuous absence of ciliated epithelium, but this is not uncommon in the area where the biopsies were taken. It is to be hoped that some temporal bones can be obtained later to make possible a comparison with normal data on the amount of ciliated cell tracks in the middle ear mucosa.
DEPARTMENT OF OTOLARYNGOLOGY, UNIVERSITY OF OULU,
Om.c, FINLAND.
REFERENCES I. Friedmann I: The pathology of secretory otitis media. Proc R Soc Med 56:695-699, 1963 2. Sade J: Pathology and pathogenesis of serous otitis media. Arch Otolaryngol 84:297305, 1966 3. Lim DJ, Viall J, Birck H, et al: Symposium on prophylaxis and treatment of middle ear effusions. Laryngoscope 82: 1625-1642, 1972 4. Tos M, Bak-Pedersen K: Density of mucous glands in a biopsy material of chronic secretory otitis media. Acta Otolaryngol (Stockh) 75:55-60, 1973 5. juhn SK, Huff JS, Paparella MM: Biochemical analyses of middle ear effusions. Ann Otol Rhinol Laryngol 80:347-353, 1971 6. Paiva T, Raunio V, Nousianen R: Secretory otitis media: protein and enzyme analyses. Ann Otol Rhinol Laryngol 83:35-43 (Suppl II),1974 7. Carlson LA, Lokk T: Protein studies of transudates of the middle ear. Scand J Clin Lab Invest 7:43-48, 1955
8. Paiva T, Paiva A, Dammert K: Middle ear mucosa and chronic ear disease. Arch Otolaryngol 91:50-56, 1970 9. Pearse, AGE: Histochemistry. London, J and A Churchill Ltd, 1960 10. Gundersen T, Gluck E: The middle ear mucosa in serous otitis media. Arch OtoIaryngol 96:40-44, 1972 II. Tos M, Bak-Pedersen K: New aspects in the pathogenesis of chronic secretory otitis media. Acta Otolaryngol (Stockh) 75:269270, 1973 12. Bernstein JM, Hayes ER, Ishikawa T, et al: Secretory otitis media: a histopathologic and immunochemical report. Trans Am Acad Ophthalmol Otolaryngol 76:1305-1318, 1972 13. Mogi G, Hon]o S, Maeda S, et al: Secretory immunoglobulin A (SIgA) in middle ear effusions. Ann Otol Rhinol Laryngol 82:302-310, 1973 14. Tender 0, Gundersen T: Nature of the fluid in serous otitis media. Arch Otolaryngol 93:473-478, 1971
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