ROLE OF PERILYMPH IN THE EARLY STAGE OF SEROUS OTITIS WOLFGANG
J.
ARNOLD,
M.D.
FRANKFORT, WEST GERMANY
SUMMARY - After injection of an electron-dense tracer into the cerebrospinal fluid (CSF) the particles can be seen within the lymphatic spaces of the middle ear mucosa after a few minutes. They find their way via the well known communication routes from CSF to the perilymphatic spaces of the inner ear. From there they enter through open fluid spaces into the fibrocytic network of the round window membrane which stands in open relationship to the extracellular fluid spaces of the middle ear mucosa. They were drained to the regional lymph nodes by lymphatic vessels. When inducing a serous otitis by experimental obstruction of the Eustachian tube it could be demonstrated that the fluid within the middle ear cavity is partly coming from the CSF and perilymph. Later the healing process of the middle ear epithelium was studied after the onset of serous otitis with and without artificial ventilation of the middle ear.
Serous otitis and the difficulty in achieving a response to therapy play a great role in the day-to-day practice of the otorhinolaryngologist. Only too frequently one finds no lasting success, in spite of numerous measures such as adenoidectomy, paracentesis, the insertion of a drain tube, or medication. A profound knowledge of the pathogenesis of this disease is required and much experimental work has been done on this subject. Until now the opinion was that hindered tube functioning causes changes in the hydrostatic pressure conditions of the middle ear, so that the epithelium is stimulated to increase "secretion." Secretion is only possible in topographic regions where secretory cell elements usually are present. If secretory active cells are absent another mechanism must be responsible for the fluid production in serous otitis. Paparella et all pointed to the participation of a vascular transudate as a source of serous fluid in the early stage of experimental serous otitis. Arnoldv" demonstrated the close relationship between cerebrospinal fluid (CSF), perilymph and the lymphatics of the middle ear mucosa. That this postulate works in humans too was shown by Vosteen and Arnold' while studying temporal bones from patients
who died from an acute cerebral hemorrhage and whose middle ear mucus layer was stained with hemosiderin. For this present study, topographic regions of the guinea pig middle ear were chosen where normally no secretory cells can be seen: the round window membrane, the adjacent mucous lining of the cochlea and the floor of the tympanic bulla. Changes of the cellular pattern therefore must be secondary to the experimental procedure. METHODS AND MATERIALS
In group A the Eustachian tubes of 18 guinea pigs, weighing between 200 and 400 g, were cauterized. In group B an additional eight animals were treated similarly after perforation of the homolateral eardrum. After 5, 10, 15, 20, 25 and 60 hours three animals were killed under anesthesia by vital perfusion with 6.4% glutaraldehyde for 30 minutes; immediately before the perfusion was performed 50 pI of thoriumdioxide were applied by the method described in a previous paper' into the medullar cistern cerebello. The temporal bones were then fixed in 1% phosphate buffered osmium tetroxide for two hours. Following a graded alcohol dehydration, the bones were dissected, while immersed in 70% alcohol under the microscope. The round window membranes and pieces of the mucosal lining from adjacent regions (floor of the bulla, antrum, mucosal cover of the cochlea, stapes) were removed and embedded in epoxy resin. Thick and thin sec-
From the ENT University Clinic of Frankfort, Frankfort, West Germany.
73
74
WOLFGANG ]. ARNOLD
B
tions were prepared for light and electron microscopic study. Thin sections were then stained with uranyl acetate; staining with lead citrate was eliminated for better detection of the thorotrast particles. For comparison, samples of middle ear mucosa from patients suffering from serous otitis were taken from the promontory during myringotomy and prepared as described. RESULTS
After sealing the tube, we injected 50 ILl thorotrast into the CSF at intervals of 5-60 hours and studied the behavior of the subepithelial fluid of the middle ear mucosa with the aid of this contrast medium. Initially, increasing hydrostatic pressure caused an enormous increase in the volume of the extracellular spaces as far as the zonulae occludens. We noted an increased cytopemptic absorbance of the fluid by the cytoplasm of the fibrocytes and the epithelial cells (Fig. 1). Under the continued influence of the vacuum in the tympanic cavity, the epithelial cells grow flatter and thinner, so that often the cytoplasmatic bridge separating the fluid of the subepithelial space from the lumen of the middle ear measures only a few thousand Angstrom. The basement membrane suffers the most damage; at some points it has double contours, at others, it is already completely broken down. Ten to 20 hours after the tubal obstruction the arrangement of the collagenous and elastic fibers of the subepithelial space is no longer parallel to the surface as is normally found, but pointing in bunches towards the surface of Fig. 1. (Top) Transmission electron micrograph of the normal round window membrane. B - Bulla tympanica. E Epithelium. F - Fibrocyte. L - Lymphatic spaces. T - Endosteal layer of scala tympani. Arrow - Basement membrane. (Approx. 5,OOOx.) (Middle) Round window membrane ten hours after tubal obstruction, three minutes after intrathecal injection of thorotrast (arrow). B - Bulla tympanica. E - Epithelium. Ly - Lymphatic spaces. F - Fibrocyte. T - Endosteal layer of scala tympani (P). (Approx. 5,OOOx.) (Lower) Epithelial layer of the mucous membrane covering the cochlea ten hours after tubal closure, three minutes after intrathecal injection of thorotrast ( arrow) . P - Pinocytotic vesicle. Arrow - Basement membrane. (Approx. 7,OOOx.)
75
PERILYMPH IN SEROUS OTITIS
..
Fig. 3. Fifteen hours after tubal obstruction. B - Bulla tympanica. Ly Lymphatic spaces. Arrow - Cytopemptic vesicle. x - Fingerlike protrusions. (Approx. 6,OOOx.)
the epithelium (Fig. 2). At other locations of the same microscopic section, the epithelial cells attempt to maintain continuity by forming finger-like cytoplasmatic appendices (Fig. 3). If at this point one investigates the behavior of the blood capillaries of the subepithelium, one finds characteristic changes here, too. The surrounding basement membrane of some capillaries disappears as the hydrostatic pressure continues to increase (10-20 hours), the cytoplasm of the vascular endothelium shows vesicles containing contrast medium from the surrounding extracellular space and, finally, one can find thorotrast particles within the lumina of the vessels themselves. In normal animals without tubal obstruction a transport of tracer from the extracellular space into capillaries or reverse was never seen! After injecting thorotrast (for control of the capillary permeability) into the carotid artery we found, at the earliest, 40-45 hours after tubal obstruction thorotrast also escapes from the capillary lumen into the extracellular space (transudate!) (Fig. 4). Twenty to 30 hours after tube closure, the epithelial structure is no longer ab~e to withstand fluid pressure, and the epithelial terminal bars separate. The fluid
then penetrates into the lumen of the tympanic cavity and the first stage of serous otitis media has been reached (Fig. 5). As subepithelial fluid begins to flow into the tympanic cavity the epitheli~m immediately attempts to effect repalfS and a new type of very dark cells is observed on the surface as early as 25 hours after tube closure. These cells extend numerous, long ramified appendices. in a basal direction into the gaps which have formed in the epithelium. A noteworthy characteristic of these cells is that they are capable of forming basement membranes and connecting with neighboring cells by means of terminal bars. The ground cytoplasm of these cells contain peculiar drop-shaped inclusions which are similar to the droplets of secretion usually found in the cytoplasm of mucus producing cells. They have at the tympanal cell surface rigid microvilli which extend unusually far into the lumen of the tympanic cavity (Fig. 6). Sixty hours after tubal obstruction one already finds a completely new type of epithelium; this is, however, not flat but consists of highly prismatic, ciliated and mucus producing cells. In many locations this metaplasia has not yet finished so that one can find all stages of epithelial changes. Now, a passage of lymph or transudate through the epithelial layer seems to be impossible. Thorotrast will not pass the basement membrane at
76
WOLFGANG ]. ARNOLD
Fig. 4. Blood capillary 45 hours after tubal obstruction, 15 minutes after intravasal injection of thorotrast. Twenty hours after the onset of middle ear effusion the capillaries show first a free passage of serous fluid into the extracellular space. Arrow - Thorotrast. Ey - Erythrocyte. Bm - Basement membrane. N - Unmyelinated nerves. F - Fibrocyte. (Approx. 24,OOOx.)
this time (Fig. 7). There are, however, areas, particularly in the vicinity of the round window membrane, where the continuity of the mucous lining surface has been completely disintegrated. Here, subepithelial fluid can still flow freely into the lumen of the tympanic cavity. It seems, indeed, that among all middle ear regions, the round window area alters most, for here one finds extensive damage, often even free erythrocytes in widened extracellular spaces (Fig. 7B).
Fig. 5. Twenty-five hours after tubal obstruction, the intercellular gaps are ruptured and the subepithelial fluid runs into the lumen (L) of the tympanic cavity. Arrow - Thorotrast. x - Basement membrane. F - Fibrocyte. (Approx. 6,OOOx.)
It must, however, be emphasized that if a large hole is punched in the tympanic membrane before tube closure, absolutely no changes occur in the epithelium! Finally, we investigated the repair processes which take place in the mucous lining of the middle ear when a large central perforation is made in the tympanic membrane after a serous otitis has begun. We found that if de-
77
PERILYMPH IN SEROUS OTITIS
Fig. 6 Twenty-five to 30 hours after the beginning of the experiment, a new type of dark cells with features of secretory cells (arrow) fills the wide intercellular gaps. Arrow - Basement membrane of the new cell type. T - Thorotrast. L -Tympanic cavity. E - Original epithelial cells. (Approx. 9,OOOx.)
II
•
•
Fig. 7. A) Forty-eight hours after tubal obstruction, repair is going on. The intercellular gaps are closed by a new type epithelium with secretory potency (arrow). Arrow - Basement membrane. F - Fibrocyte. T - Thorotrast. (Approx. 8,OOOx.)
78
WOLFGANG ]. ARNOLD
Fig. 7. B) The new metaplastic epithelium covers the cochlea near the round window membrane. Now it is very difficult to distinguish it from the epithelium near the tubal orifice. K - Collagen.
struction of the tympanic membrane was done before metaplastic changes of the epithelial cells occurred (up to 25 hours), the epithelium recovers completely to its normal flat pattern. Once metaplasia has started no return to the original cell type could be observed after ventilation. DISCUSSION
The results of this study open a new aspect of understanding the source of water-like fluid in serous otitis. Most of the investigations concerning this problem are fixed on the hypothesis that the fluid must be a product of cells or a capillary transudate. Our study on the significance qf the subepithelial space
of the middle ear mucosa2 demonstrated clearly that most of the extracellular fluid of the submucosa is lymph which stands in direct connection to the perilymph of scala tympani. Arnold and v. IlbergG,7 described the pathways of this connection and found the round window to be the most important conduit to the lymphatics of the middle ear mucosa. Therefore it was not surprising to see this fluid within the tympanic cavity after artificially producing hydrostatic pressure in the middle ear by tube closure. The morphology of the normal mucous lining of the guinea pig middle ear gives rise to a number of fundamental
PERILYMPH IN SEROUS OTITIS
7'9
questions. Usually, one finds highly prismatic epithelium with kinocilia and, in between, goblet cells only in the region of the ostium of the tympanic tube,"?" whereas most parts of the mesotympanum show a flat, squamous epithelium. The histology of the round window membrane was first described by Hoft,t2 Kawabata and Paparella," and Arnold," confirming these observations; the membrane as well as the neighboring mucosa is always covered by a flat epithelium without kinocilia or mucus producing cells. This part was an excellent model to study changes under hydrostatic conditions. Lim et a1/ 4 and Hussl and Lim-! have already demonstrated the mode of secretion very well by electron microscopic methods. But they performed their studies in topographic regions of the middle ear where mucous glands are normally present, near the tympanal tube orifice.
the subepithelial space reveals capillarylike formations consisting of fibrocytic appendices, whose lumina contain thorotrast even 30 seconds after injection into the CSF. These are lymphatic vessels which serve practically as the drainage system of the fibrocytic sponge." It was not surprising that contrast medium particles like thorotrast or india ink were found in the regional lymph nodes two to three minutes after intrathecal injection." In serous otitis this fluid is pressed to the surface. In the early stage of serous otitis, lymph is the main component of the fluid in the tympanic cavity. Later, a real metaplasia occurs and creates a new epithelial surface with mucus producing cells. With this new aspect we want to draw the attention to a physiological fact which could be important not only for the understanding of serous otitis but also for other ear diseases.1 5 , 1 6
If one remembers, however, that the major portion of the mucous lining of the middle ear consists of flat epithelium without goblet cells and kinocilia, one realizes that there must be another process which insures constant moistening of the mucous membrane surface. The morphologist notes that the cytoplasm of the flat epithelial cells contains many optically empty vesicles which can be seen as far as the cell surface. The subepithelial space itself consists of a loose network of fibrocytes whose mesh contains collagenic and elastic fibers. Apart from this, there is only an optically empty space. Optically empty spaces in morphology must be interpreted as "fluid." We showed that this fluid consists of lymph which is supplied from CSF and perilymph. A thorough study of the structures of
Finally, one must note that, in serous otitis, the morphology of mucosal sampIes taken from the promontory of adult patients show the same structure as could be seen in this experiment, dilated epithelium with wide open intercellular gaps, but mostly no secretory cell elements. It is a very interesting fact that children mostly demonstrate a mucotympanum with a completely metaplastic epithelium (secretory cells, kinocilia, etc. ); whereas, in adult patients, mostly a serotympanum is diagnosed. This observation is very difficult to interpret but it seems that it takes a long time to recognize the hearing loss in children, whereas adults note a reduced hearing capacity very soon and, therefore, therapy is started earlier than for children, where metaplasia of the epithelium has already begun.17.l8
REFERENCES 1. Paparella MM, Hiraide F, Juhn S, et 4. Vosteen KH, Arnold W: Hi:irsturz nach al, Cellular events involved in middle ear Blutung aus einem Basilarisaneurysma. Arch fluid production. Ann Otol Rhinol Laryngol Klin Exp Ohren Nasen Kehlkopfheilkd, to be 79:766-780, 1970 published 2. Arnold W: Die Bedeutung des sub5. Arnold W, Nitze HR, Ritter R, et al: epithelial en Raumes der Mittelohrschleimhaut. Qualitative Untersuchungen der VerbindungsArch Klin Exp Ohren Nasen Kehlkopfhielkd wege des Subarachnoidalraumes mit dem 198:262-280, 1971 lymphatischen System des Kopfes und des 3. Arnold W: Zur Frage der Produktion Halses. Acta Otolaryngol (Stockh) 74:411und Resorption der Perilymphe, Z Laryng 424, 1972 Rhinol Otol 53:774-790, 1974 6. Arnold W, von Ilberg C: Verbindungs-
80
WOLFGANG ]. ARNOLD
wege zwischen Liquor und Perilymphraum. Arch Klin Exp Ohren Nasen Kehlkopfheilkd 198:247-261, 1971 7. Arnold W, von Ilberg C: Neue Aspekte zur Morphologie und Funktion des runden Fensters. Z Laryng Rhinol Otol 51:390-399, 1972 8. Arnold W: Ultrastrukturelle experimentelle Untersuchungen zur Pathogenese des serosen Paukenergusses. Arch Klin Exp Ohren Nasen Kehlkopfheilkd 201 :91-107, 1972 9. Haye R: The epithelium of the middle ear in the guinea pig. Z Zellforsch 138:283~ 294, 1973 10. Lim DJ, Hussl B: Tympanic mucosa after tubal obstruction. An ultrastructural observation. Arch Otolaryngol 91 :585-593, 1970 11. Hussl B, Lim DJ: Driisenzellen in normaler tierischer und menschlicher Mittelohrschleimhaut. Arch Klin Exp Ohren Nasen Kehlkopfheilkd 193:337-350, 1969 12. HOft J: Die Permeabilltat und die Beeinflussung der Permeabilitat der Membran des runden Fensters durch Pantocain (Tetra-
cain). Arch Klin Exp Ohren Nasen Kehlkopfheilkd 193:128-137, 1969 13. Kawabata J, Paparella MM: Ultrastructure of normal human middle ear mucosa. Ann Otol Rhinol Laryngol 78:125-137, 1969 14. Lim DJ, Paparella MM, Kimura RS: Ultrastructure of the Eustachian tube and middle ear mucosa in the guinea pig. Acta Otolaryngol (Stockh) 63:425-444, 1967 15. Goodhill V, Harris I, Brockman SJ, et al, Sudden deafness and labyrinthine window ruptures. Ann Otol Rhinol Laryngol 82:2-12, 1973 16. Farrior JB, Endicott IN: Congenital mixed deafness: Cerebrospinal fluid otorrhea. Ablation of the aqueduct of the cochlea. Laryngoscope 81: 684-693, 1971 17. PIester D, Arnold W, Opitz HJ: Das Seromucotympanon. Arch Klin Exp Ohren Nasen Kehlkopfheilkd 207:527-535, 1974 18. Arnold W, Vosteen KH: Die Reaktion der Mittelohrschleimhaut bei Tubenverschlu,8. Acta Otolaryngol [Suppl] (Stockh) 330:4863, 1975
REPRINTS - Wolfgang Arnold, M.D., ENT-University Clinic, 6 Frankfort 70, TheodorStern-Kai 7, Frankfort, West Germany. ACKNOWLEDGEMENTS - S. Linnenkohl aided in the study.
J.
ARNOLD,
M.D.
FRANKFORT, WEST GERMANY
SUMMARY - After injection of an electron-dense tracer into the cerebrospinal fluid (CSF) the particles can be seen within the lymphatic spaces of the middle ear mucosa after a few minutes. They find their way via the well known communication routes from CSF to the perilymphatic spaces of the inner ear. From there they enter through open fluid spaces into the fibrocytic network of the round window membrane which stands in open relationship to the extracellular fluid spaces of the middle ear mucosa. They were drained to the regional lymph nodes by lymphatic vessels. When inducing a serous otitis by experimental obstruction of the Eustachian tube it could be demonstrated that the fluid within the middle ear cavity is partly coming from the CSF and perilymph. Later the healing process of the middle ear epithelium was studied after the onset of serous otitis with and without artificial ventilation of the middle ear.
Serous otitis and the difficulty in achieving a response to therapy play a great role in the day-to-day practice of the otorhinolaryngologist. Only too frequently one finds no lasting success, in spite of numerous measures such as adenoidectomy, paracentesis, the insertion of a drain tube, or medication. A profound knowledge of the pathogenesis of this disease is required and much experimental work has been done on this subject. Until now the opinion was that hindered tube functioning causes changes in the hydrostatic pressure conditions of the middle ear, so that the epithelium is stimulated to increase "secretion." Secretion is only possible in topographic regions where secretory cell elements usually are present. If secretory active cells are absent another mechanism must be responsible for the fluid production in serous otitis. Paparella et all pointed to the participation of a vascular transudate as a source of serous fluid in the early stage of experimental serous otitis. Arnoldv" demonstrated the close relationship between cerebrospinal fluid (CSF), perilymph and the lymphatics of the middle ear mucosa. That this postulate works in humans too was shown by Vosteen and Arnold' while studying temporal bones from patients
who died from an acute cerebral hemorrhage and whose middle ear mucus layer was stained with hemosiderin. For this present study, topographic regions of the guinea pig middle ear were chosen where normally no secretory cells can be seen: the round window membrane, the adjacent mucous lining of the cochlea and the floor of the tympanic bulla. Changes of the cellular pattern therefore must be secondary to the experimental procedure. METHODS AND MATERIALS
In group A the Eustachian tubes of 18 guinea pigs, weighing between 200 and 400 g, were cauterized. In group B an additional eight animals were treated similarly after perforation of the homolateral eardrum. After 5, 10, 15, 20, 25 and 60 hours three animals were killed under anesthesia by vital perfusion with 6.4% glutaraldehyde for 30 minutes; immediately before the perfusion was performed 50 pI of thoriumdioxide were applied by the method described in a previous paper' into the medullar cistern cerebello. The temporal bones were then fixed in 1% phosphate buffered osmium tetroxide for two hours. Following a graded alcohol dehydration, the bones were dissected, while immersed in 70% alcohol under the microscope. The round window membranes and pieces of the mucosal lining from adjacent regions (floor of the bulla, antrum, mucosal cover of the cochlea, stapes) were removed and embedded in epoxy resin. Thick and thin sec-
From the ENT University Clinic of Frankfort, Frankfort, West Germany.
73
74
WOLFGANG ]. ARNOLD
B
tions were prepared for light and electron microscopic study. Thin sections were then stained with uranyl acetate; staining with lead citrate was eliminated for better detection of the thorotrast particles. For comparison, samples of middle ear mucosa from patients suffering from serous otitis were taken from the promontory during myringotomy and prepared as described. RESULTS
After sealing the tube, we injected 50 ILl thorotrast into the CSF at intervals of 5-60 hours and studied the behavior of the subepithelial fluid of the middle ear mucosa with the aid of this contrast medium. Initially, increasing hydrostatic pressure caused an enormous increase in the volume of the extracellular spaces as far as the zonulae occludens. We noted an increased cytopemptic absorbance of the fluid by the cytoplasm of the fibrocytes and the epithelial cells (Fig. 1). Under the continued influence of the vacuum in the tympanic cavity, the epithelial cells grow flatter and thinner, so that often the cytoplasmatic bridge separating the fluid of the subepithelial space from the lumen of the middle ear measures only a few thousand Angstrom. The basement membrane suffers the most damage; at some points it has double contours, at others, it is already completely broken down. Ten to 20 hours after the tubal obstruction the arrangement of the collagenous and elastic fibers of the subepithelial space is no longer parallel to the surface as is normally found, but pointing in bunches towards the surface of Fig. 1. (Top) Transmission electron micrograph of the normal round window membrane. B - Bulla tympanica. E Epithelium. F - Fibrocyte. L - Lymphatic spaces. T - Endosteal layer of scala tympani. Arrow - Basement membrane. (Approx. 5,OOOx.) (Middle) Round window membrane ten hours after tubal obstruction, three minutes after intrathecal injection of thorotrast (arrow). B - Bulla tympanica. E - Epithelium. Ly - Lymphatic spaces. F - Fibrocyte. T - Endosteal layer of scala tympani (P). (Approx. 5,OOOx.) (Lower) Epithelial layer of the mucous membrane covering the cochlea ten hours after tubal closure, three minutes after intrathecal injection of thorotrast ( arrow) . P - Pinocytotic vesicle. Arrow - Basement membrane. (Approx. 7,OOOx.)
75
PERILYMPH IN SEROUS OTITIS
..
Fig. 3. Fifteen hours after tubal obstruction. B - Bulla tympanica. Ly Lymphatic spaces. Arrow - Cytopemptic vesicle. x - Fingerlike protrusions. (Approx. 6,OOOx.)
the epithelium (Fig. 2). At other locations of the same microscopic section, the epithelial cells attempt to maintain continuity by forming finger-like cytoplasmatic appendices (Fig. 3). If at this point one investigates the behavior of the blood capillaries of the subepithelium, one finds characteristic changes here, too. The surrounding basement membrane of some capillaries disappears as the hydrostatic pressure continues to increase (10-20 hours), the cytoplasm of the vascular endothelium shows vesicles containing contrast medium from the surrounding extracellular space and, finally, one can find thorotrast particles within the lumina of the vessels themselves. In normal animals without tubal obstruction a transport of tracer from the extracellular space into capillaries or reverse was never seen! After injecting thorotrast (for control of the capillary permeability) into the carotid artery we found, at the earliest, 40-45 hours after tubal obstruction thorotrast also escapes from the capillary lumen into the extracellular space (transudate!) (Fig. 4). Twenty to 30 hours after tube closure, the epithelial structure is no longer ab~e to withstand fluid pressure, and the epithelial terminal bars separate. The fluid
then penetrates into the lumen of the tympanic cavity and the first stage of serous otitis media has been reached (Fig. 5). As subepithelial fluid begins to flow into the tympanic cavity the epitheli~m immediately attempts to effect repalfS and a new type of very dark cells is observed on the surface as early as 25 hours after tube closure. These cells extend numerous, long ramified appendices. in a basal direction into the gaps which have formed in the epithelium. A noteworthy characteristic of these cells is that they are capable of forming basement membranes and connecting with neighboring cells by means of terminal bars. The ground cytoplasm of these cells contain peculiar drop-shaped inclusions which are similar to the droplets of secretion usually found in the cytoplasm of mucus producing cells. They have at the tympanal cell surface rigid microvilli which extend unusually far into the lumen of the tympanic cavity (Fig. 6). Sixty hours after tubal obstruction one already finds a completely new type of epithelium; this is, however, not flat but consists of highly prismatic, ciliated and mucus producing cells. In many locations this metaplasia has not yet finished so that one can find all stages of epithelial changes. Now, a passage of lymph or transudate through the epithelial layer seems to be impossible. Thorotrast will not pass the basement membrane at
76
WOLFGANG ]. ARNOLD
Fig. 4. Blood capillary 45 hours after tubal obstruction, 15 minutes after intravasal injection of thorotrast. Twenty hours after the onset of middle ear effusion the capillaries show first a free passage of serous fluid into the extracellular space. Arrow - Thorotrast. Ey - Erythrocyte. Bm - Basement membrane. N - Unmyelinated nerves. F - Fibrocyte. (Approx. 24,OOOx.)
this time (Fig. 7). There are, however, areas, particularly in the vicinity of the round window membrane, where the continuity of the mucous lining surface has been completely disintegrated. Here, subepithelial fluid can still flow freely into the lumen of the tympanic cavity. It seems, indeed, that among all middle ear regions, the round window area alters most, for here one finds extensive damage, often even free erythrocytes in widened extracellular spaces (Fig. 7B).
Fig. 5. Twenty-five hours after tubal obstruction, the intercellular gaps are ruptured and the subepithelial fluid runs into the lumen (L) of the tympanic cavity. Arrow - Thorotrast. x - Basement membrane. F - Fibrocyte. (Approx. 6,OOOx.)
It must, however, be emphasized that if a large hole is punched in the tympanic membrane before tube closure, absolutely no changes occur in the epithelium! Finally, we investigated the repair processes which take place in the mucous lining of the middle ear when a large central perforation is made in the tympanic membrane after a serous otitis has begun. We found that if de-
77
PERILYMPH IN SEROUS OTITIS
Fig. 6 Twenty-five to 30 hours after the beginning of the experiment, a new type of dark cells with features of secretory cells (arrow) fills the wide intercellular gaps. Arrow - Basement membrane of the new cell type. T - Thorotrast. L -Tympanic cavity. E - Original epithelial cells. (Approx. 9,OOOx.)
II
•
•
Fig. 7. A) Forty-eight hours after tubal obstruction, repair is going on. The intercellular gaps are closed by a new type epithelium with secretory potency (arrow). Arrow - Basement membrane. F - Fibrocyte. T - Thorotrast. (Approx. 8,OOOx.)
78
WOLFGANG ]. ARNOLD
Fig. 7. B) The new metaplastic epithelium covers the cochlea near the round window membrane. Now it is very difficult to distinguish it from the epithelium near the tubal orifice. K - Collagen.
struction of the tympanic membrane was done before metaplastic changes of the epithelial cells occurred (up to 25 hours), the epithelium recovers completely to its normal flat pattern. Once metaplasia has started no return to the original cell type could be observed after ventilation. DISCUSSION
The results of this study open a new aspect of understanding the source of water-like fluid in serous otitis. Most of the investigations concerning this problem are fixed on the hypothesis that the fluid must be a product of cells or a capillary transudate. Our study on the significance qf the subepithelial space
of the middle ear mucosa2 demonstrated clearly that most of the extracellular fluid of the submucosa is lymph which stands in direct connection to the perilymph of scala tympani. Arnold and v. IlbergG,7 described the pathways of this connection and found the round window to be the most important conduit to the lymphatics of the middle ear mucosa. Therefore it was not surprising to see this fluid within the tympanic cavity after artificially producing hydrostatic pressure in the middle ear by tube closure. The morphology of the normal mucous lining of the guinea pig middle ear gives rise to a number of fundamental
PERILYMPH IN SEROUS OTITIS
7'9
questions. Usually, one finds highly prismatic epithelium with kinocilia and, in between, goblet cells only in the region of the ostium of the tympanic tube,"?" whereas most parts of the mesotympanum show a flat, squamous epithelium. The histology of the round window membrane was first described by Hoft,t2 Kawabata and Paparella," and Arnold," confirming these observations; the membrane as well as the neighboring mucosa is always covered by a flat epithelium without kinocilia or mucus producing cells. This part was an excellent model to study changes under hydrostatic conditions. Lim et a1/ 4 and Hussl and Lim-! have already demonstrated the mode of secretion very well by electron microscopic methods. But they performed their studies in topographic regions of the middle ear where mucous glands are normally present, near the tympanal tube orifice.
the subepithelial space reveals capillarylike formations consisting of fibrocytic appendices, whose lumina contain thorotrast even 30 seconds after injection into the CSF. These are lymphatic vessels which serve practically as the drainage system of the fibrocytic sponge." It was not surprising that contrast medium particles like thorotrast or india ink were found in the regional lymph nodes two to three minutes after intrathecal injection." In serous otitis this fluid is pressed to the surface. In the early stage of serous otitis, lymph is the main component of the fluid in the tympanic cavity. Later, a real metaplasia occurs and creates a new epithelial surface with mucus producing cells. With this new aspect we want to draw the attention to a physiological fact which could be important not only for the understanding of serous otitis but also for other ear diseases.1 5 , 1 6
If one remembers, however, that the major portion of the mucous lining of the middle ear consists of flat epithelium without goblet cells and kinocilia, one realizes that there must be another process which insures constant moistening of the mucous membrane surface. The morphologist notes that the cytoplasm of the flat epithelial cells contains many optically empty vesicles which can be seen as far as the cell surface. The subepithelial space itself consists of a loose network of fibrocytes whose mesh contains collagenic and elastic fibers. Apart from this, there is only an optically empty space. Optically empty spaces in morphology must be interpreted as "fluid." We showed that this fluid consists of lymph which is supplied from CSF and perilymph. A thorough study of the structures of
Finally, one must note that, in serous otitis, the morphology of mucosal sampIes taken from the promontory of adult patients show the same structure as could be seen in this experiment, dilated epithelium with wide open intercellular gaps, but mostly no secretory cell elements. It is a very interesting fact that children mostly demonstrate a mucotympanum with a completely metaplastic epithelium (secretory cells, kinocilia, etc. ); whereas, in adult patients, mostly a serotympanum is diagnosed. This observation is very difficult to interpret but it seems that it takes a long time to recognize the hearing loss in children, whereas adults note a reduced hearing capacity very soon and, therefore, therapy is started earlier than for children, where metaplasia of the epithelium has already begun.17.l8
REFERENCES 1. Paparella MM, Hiraide F, Juhn S, et 4. Vosteen KH, Arnold W: Hi:irsturz nach al, Cellular events involved in middle ear Blutung aus einem Basilarisaneurysma. Arch fluid production. Ann Otol Rhinol Laryngol Klin Exp Ohren Nasen Kehlkopfheilkd, to be 79:766-780, 1970 published 2. Arnold W: Die Bedeutung des sub5. Arnold W, Nitze HR, Ritter R, et al: epithelial en Raumes der Mittelohrschleimhaut. Qualitative Untersuchungen der VerbindungsArch Klin Exp Ohren Nasen Kehlkopfhielkd wege des Subarachnoidalraumes mit dem 198:262-280, 1971 lymphatischen System des Kopfes und des 3. Arnold W: Zur Frage der Produktion Halses. Acta Otolaryngol (Stockh) 74:411und Resorption der Perilymphe, Z Laryng 424, 1972 Rhinol Otol 53:774-790, 1974 6. Arnold W, von Ilberg C: Verbindungs-
80
WOLFGANG ]. ARNOLD
wege zwischen Liquor und Perilymphraum. Arch Klin Exp Ohren Nasen Kehlkopfheilkd 198:247-261, 1971 7. Arnold W, von Ilberg C: Neue Aspekte zur Morphologie und Funktion des runden Fensters. Z Laryng Rhinol Otol 51:390-399, 1972 8. Arnold W: Ultrastrukturelle experimentelle Untersuchungen zur Pathogenese des serosen Paukenergusses. Arch Klin Exp Ohren Nasen Kehlkopfheilkd 201 :91-107, 1972 9. Haye R: The epithelium of the middle ear in the guinea pig. Z Zellforsch 138:283~ 294, 1973 10. Lim DJ, Hussl B: Tympanic mucosa after tubal obstruction. An ultrastructural observation. Arch Otolaryngol 91 :585-593, 1970 11. Hussl B, Lim DJ: Driisenzellen in normaler tierischer und menschlicher Mittelohrschleimhaut. Arch Klin Exp Ohren Nasen Kehlkopfheilkd 193:337-350, 1969 12. HOft J: Die Permeabilltat und die Beeinflussung der Permeabilitat der Membran des runden Fensters durch Pantocain (Tetra-
cain). Arch Klin Exp Ohren Nasen Kehlkopfheilkd 193:128-137, 1969 13. Kawabata J, Paparella MM: Ultrastructure of normal human middle ear mucosa. Ann Otol Rhinol Laryngol 78:125-137, 1969 14. Lim DJ, Paparella MM, Kimura RS: Ultrastructure of the Eustachian tube and middle ear mucosa in the guinea pig. Acta Otolaryngol (Stockh) 63:425-444, 1967 15. Goodhill V, Harris I, Brockman SJ, et al, Sudden deafness and labyrinthine window ruptures. Ann Otol Rhinol Laryngol 82:2-12, 1973 16. Farrior JB, Endicott IN: Congenital mixed deafness: Cerebrospinal fluid otorrhea. Ablation of the aqueduct of the cochlea. Laryngoscope 81: 684-693, 1971 17. PIester D, Arnold W, Opitz HJ: Das Seromucotympanon. Arch Klin Exp Ohren Nasen Kehlkopfheilkd 207:527-535, 1974 18. Arnold W, Vosteen KH: Die Reaktion der Mittelohrschleimhaut bei Tubenverschlu,8. Acta Otolaryngol [Suppl] (Stockh) 330:4863, 1975
REPRINTS - Wolfgang Arnold, M.D., ENT-University Clinic, 6 Frankfort 70, TheodorStern-Kai 7, Frankfort, West Germany. ACKNOWLEDGEMENTS - S. Linnenkohl aided in the study.
