Acta Oto-Laryngologica
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Damage to reissner's membrane in the guinea-pig cochlea following acute atoxyl intoxication M. Anniko To cite this article: M. Anniko (1976) Damage to reissner's membrane in the guinea-pig cochlea following acute atoxyl intoxication, Acta Oto-Laryngologica, 81:5-6, 415-423, DOI: 10.3109/00016487609107495 To link to this article: http://dx.doi.org/10.3109/00016487609107495
Published online: 08 Jul 2009.
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Date: 21 March 2016, At: 18:13
Acta Otolaryngol81: 415-423, 1976
DAMAGE TO REISSNER’S MEMBRANE IN THE GUINEA-PIG COCHLEA FOLLOWING ACUTE ATOXYL INTOXICATION M. Anniko Frorii the Deppcrrtnirtit of Otoltintigolog.v, Huddinge Sjuklius, the Krrrolinskri Institute. cind the King Gustin>V Resecirch Institute. the KurolitisXir Institute, Stockholm, Stt‘eden
Downloaded by [University of New England] at 18:13 21 March 2016
(Received August 28, 1975)
ing endolymph and perilymph is minimal. It may therefore play an important role in maintaining the difference in composition between the two fluids (Iurato, 1967). The relative proportions of electrolytes in the endolymph are quite different from those in the perilymph (Smith et al., 1954; Citron et al., 1956; Rauch & Kostlin, 1958). Two hypotheses have been advanced to explain how this difference in composition is maintained. According to the “longitudinal-flow’’ theory The ultrastructure of Reissner’s membrane (Corti, 1851; Guild, 1927; Lundquist, 1965) has been described in a number of publica- the endolymph is secreted in the stria vascutions (Hagiwara, 1963; Duvall & Rhodes, laris, runs along the cochlear duct and is re1967; Watanuki, 1968; Iurato & Taidelli, 1967; absorbed in the endolymphatic sac. This Duvall & Sutherland, 1970). The membrane is theory, which is supported by Tasaki et al. situated between the spiral limbus and the (1954) and Legouix (1962), also implies that vestibular crest of the spiral ligament; it is 2-3 Reissner’s membrane is practically impermenm thick, and consists of two layers. Towards able to sodium and potassium ions. On the the endolymphatic space there is a simple other hand, Naftalin & Harrison (1958), squamous epithelium. The surface facing the Lawrence et al. (1961) and Rauch et al. scala vestibuli consists of a layer of flat meso- (1963) support the “radial-flow’’ theory, i.e. thelial cells. A distinct basement membrane the hypothesis that the endolymph is formed is interposed between the two cell layers from peril ymph by passing through Reissner’s (Iurato & Taidelli, 1971). Reissner’s mem- membrane. According to this theory, the brane represents a portion of the membranous function of the stria vascularis is mainly ablabyrinth where the amount of tissue separat- sorptive. Compared to other tissues, the respiratory Supported by grants from the Karolinska Institute and rate of Reissner’s membrane is very high from the Swedish Medical Research Council (grant no. (Chou, 1963). Watanuki et al. (1968) showed 12X-720). Ahstroct. The ototoxic effects of the arsenic compound atoxyl has been earlier demonstrated by Anniko & Wersall (1975) in the stria vascularis of the guinea pig cochlea. The aim of the present investigation was to study the morphological degeneration at the ultrastructural level after damaging the Reissner’s membrane experimentally. I n acute atoxyl intoxication the first signs of degeneration appear after approximately I 2 hours. After 24 hours, the membrane may be markedly degenerated. Both the epithelial and mesothelial cells become vacuolized, the mitochondria lose their internal structure and become swollen, and lipid granules are found in the cytoplasm. Mesothelial cells are damaged to a lesser extent than the epithelial cells in the same specimen.
Acta Otolaryngol81
416 M . Anniko Table I . Administration of atoxyl to guinea pigs in the experimental group Amount injected on each occasion (mg/kg)
Animal no. ~
70 70
3 2
2 I /4
100
I
70 I40 I40
2
Total amount injected (mg/kg)
Interval between last injection and sacrifice (days)
Preyer reflex
~~~
20 52
53 54 55 63
Downloaded by [University of New England] at 18:13 21 March 2016
Number of injections
Injection period (days)
I /4
I 1
a higher turnover of nucleic acid (DNA) in the epithelial cells on the inner side of Reissner’s membrane than in the mesothelial cells on the outer side of it. Plester (1963) reported reduced protein synthesis in Reissner’s membrane after kanamycin administration. Misrahy et al. (1962) reported increased permeability after kanamycin treatment. Kaneko et al. (1970) suggest that after kanamycin administration Reissner’s membrane may lose its ability to maintain the normal chemical composition of the perilymph and endolymph. Tracers have been introduced into the scala vestibuli by many investigators (Tonndorf et al. 1962; Rudert, 1969; Duvall & Quick, 1969; von Ilberg, 1968; Rauch, 1966). There appears to be a selective passage of some dyes (methylene blue, toluidine blue and ferritin) through the membrane, while others (methyl blue, Janus green) do not pass through it (von Bekesy, 1953; Duvall & Tonndorf. 1962; Hinojosa, 1971). Nowak ( 1974) reported altered permeability of Reissner’s membrane following streptomycin administration. The aim of the present study was to investigate the effect on Reissner’s membrane of atoxyl (Pro Gen@ Sodium), an arsenic compound (sodium arsenilate) with known ototoxic properties (Anniko & Wersall. 1975). MATERIALS AND METHODS Nine healthy, young guinea pigs with a normal Preyer’s reflex, weighing around 25b.350 g were used for the experiment. Six animals Ac,fo O t i h r v n # i ~XI l
210 140
1
I /4
100
I
I40 I40 140
I /4
I I /2
were injected with sodium arsanilate (atoxyl). The control group consisted of 3 healthy, untreated guinea pigs. Each animal was injected subcutaneously with a 2 % solution of atoxyl in sterile water over a certain period of time. The amount of atoxyl injected on each occasion varied between 70 and 140 mg per kg body weight. The total dose ranged from 100-2 10 mg per kg body weight. The period of administration varied between 6 hours and 2 days and the survival time before decapitation between 6 hours and I day after the last injection (Table I). The specimens were treated according to standard methods for light and electron microscopy of osmium tetroxide-fixed specimens (Anniko & Wersall, 1975). RESULTS Structural alterations were observed as early as 12 hours after atoxyl administration. After 24-48 hours marked degeneration of Reissner’s membrane was observed. Administration of 70 mg of atoxyl per kg body weight twice within 48 hours and sacrificing the guinea pig 24 hours after the last injection resulted in very severe damage to the membrane in all coils, there being no apparent difference between the turns. Changes in all coils were observed when 70 mg of atoxyl per kg body weight was administered twice within 6 hours and the animal was sacrificed 12 hours after the first injection. The Reissner’s membrane in the basal coils in these animals was, however, less severely damaged
417
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Damage to Reissner’s membrane following atoxyl intoxication
Fig. I . The position of Reissner’s membrane is changed during the degeneration and it becomes depressed over
Fig. 2. Electron micrograph (EM). Degenerating endothelial ( E ) cell with massive vacuolization.
the organ of Corti as the damage proceeds. Light microscopy. x200. Acto Otoluryngol81
M.Anniko
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418
Fig. 3. (A) EM. Endothelial ( E )and mesothelial ( M ) cells
with mitochondria, showing fragmentation of their internal structure (arrows). (B) EM. Accumulations of electrondense material in the cytoplasm (mitochondria?) of endothelial ( E ) and mesothelial ( M ) cells (arrows). (C) EM.
Acta Otolaryngol81
Differentiation of the degenerative pattern of the two cell types in Reissner’s membrane; the electron-dense appearance of the endothelial ( E ) cell cytoplasm can be seen. (D) EM.Lipoid-like accumulations in the cytoplasm of an endothelial ( E )cell.
Downloaded by [University of New England] at 18:13 21 March 2016 Fig. 4. (A) EM. A “dark” endothelial cell ( E ) with a high degree of distortion. The remains of the mesothelial cell reveal a large vacuole facing the perilymph. (B) EM.
Accumulations of very electron-dense material in the cytoplasm of a “dark” endothelial ( E ) cell. Acta Ocolaryngol81
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420 M . Anniko
than the rest of the cochlea. Animals which received 100 mg of atoxyl per kg body weight 24 hours before sacrifice showed only minimal degeneration of the Reissner’s membrane restricted to the apical coils. The same changes were observed when 140 mg of atoxyl per kg body weight was administered and the animals were sacrificed after the same time interval. The labyrinth from animals sacrificed 12 hours after such treatment showed no morphological changes. The position of Reissner’s membrane alters depending on the degree of intoxication. In some animals with early degeneration of the membrane the whole membrane was seen to bulge out towards the scala vestibuli (hydrops labyrinthine). In severe atoxyl intoxication the membrane become depressed and lies over the tectorial membrane and the Hensen cells but seldom touches the stria vascularis (Fig. I). All the cells within Reissner’s membrane were affected, the most severe degeneration being regularly found in the apical coils. Lesions occur both in the insertion of the membrane at the stria vascularis and in the area of the insertion at the spiral limbus. The squamous epithelial cells The squamous epithelium cells, which normally contains a moderate number of vesicles and pinocytotic vesicles, showed different degrees of vacuolization which was so widespread in some specimens that almost the whole cell was filled with vacuoles and the degenerating cell organelles are pressed towards the cell periphery (Fig. 2). The vacuolization becomes most widespread in the area around the nucleous. However, vacuoles of considerable size may also be observed in the region of the cell junction between two squamous epithelium cells. The normal cell appearance, with microvilli, tubules of the endoplasmic reticula, a small Golgi apparatus, a few microtubules and filaments and pinocytotic vesicles, is lost rather early during the degeneration process. Acta Otolaryngol81
Reductions in the numbers of microvilli and pinocytotic vesicles are the first signs of damage. The mitochondria swell and the cristae mitochondriales become fragmented (Fig. 3A). In severely damaged cells the mitochondria contain varying amounts of electrondense, osmiophilic material and this may even fill the mitochondrion completely (Fig. 3 B). Lipoid-like granules as large as 1&50 mitochondria are observed in the cytoplasm at all stages of degeneration (Fig. 3D). Some severely damaged squamous epithelium cells throughout the cytoplasm of the cell have an electron dense appearance (Fig. 3C). The cells are distorted but they still take part in the lining of Reissner’s membrane separating the two fluid compartments from each other (Fig. 4 A, B). The free surface of the epithelial cell layer towards the endolymph is uneven. However, free ribosomes, damaged mitochondria, fat granules and also accumulations of even more electron-dense material are also observed in these cells. The vacuolization is poor. The cell nucleous is resistant to degeneration and is the last cell component to disintegrate. It becomes involved in the final stage of damage by fragmentation of the chromatin and rupture of the nuclear membrane. The mesothelial cells The degeneration of the mesothelial cells follows a similar course to that of the epithelial cells. The cells become vacuolized, very few pinocytotic vesicles are observed and the internal structure of the mitochondria is fragmented but the mitochondria have fewer accumulations of electron dense material than those in the epithelial cells. Few fat granules are seen and they are not of the same size as those in the epithelial cells. Mesothelial cells with an electron-dense appearance similar to that of the cytoplasm of the whole cell have not been found. The mesothelial cells are always less degenerated than the squamous epithelial cells in the same specimen. Mesothelial cells adjacent to the scala vesti-
buli frequently contained rather large vacuoles on the cell membrane facing the perilymph. The degeneration of Reissner’s membrane starts in the apical part and it must undergo rather severe changes before its position changes. The electron-dense squamous epithelium cells were only found in animals where the Reissner’s membrane had changed from its normal position.
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DISCUSSION The present investigation reveals that atoxyl has a damaging effect on Reissner’s membrane in the guinea-pig. I n the initial phase of degeneration, no pathological changes are observed in the light microscope, while the electron microscope shows morphological alterations. Kaneko et al. (1970) reported that after kanamycin administration the epithelial cells in Reissner’s membrane showed vacuolization and condensation of cytoplasmic granules. They classified the cells into two types according to their cytoplasmic appearancelight and dark cells. The light cells showed vacuolization of the cytoplasm and little change in most of the cell organelles. On the other hand, the density of mitochondria and cell granules in the dark cells was increased. Both cell types showed decreased numbers of pinocytotic vesicles. The present results demonstrate the existence of some cells with comparatively dense cytoplasm. However, although the cell organelles in these “dark” cells are more electron dense than normal they take part in the degenerative process. It has been reported from electron microscopic studies that the epithelial cells of Reissner’s membrane contain many vesicles which may be involved in micropinocytosis. A well developed Golgi complex is found in the cytoplasm and many microvilli are observed on the endolymphatic surface (Hagiwara, 1%3; Iurato, 1967; Duvall & Modes, 1967). These findings suggest a high degree of metabolic activity.
In guinea-pigs made deaf by the administration of kanamycin, micropinocytotic vesicles were very rarely seen in the epithelial cells of Reissner’s membrane. Therefore, it is unlikely that active transport by the pinocytotic system is functional in these cells (Kaneko et al.. 1970). The two pathways from the scala vestibuli to the scala media through Reissner’s membrane via the intercellular junction and via pinocytosis have been demonstrated by Duvall & Quick (1969) and Duvall & Sutherland (1970). Misrahy et al. (1962) reported increased permeability after kanamycin administration, hypoxia and acoustic overstimulation. Nowak ( 1974) showed vacuolization. increased formation of lysosomes, lipid granules and an increased number of free ribosomes after dihydrostreptomycin administration. Fragmentation of the zonula occludents and desmosomes was also reported. Schatzle (1972) proposed that the lipid granules were formed by damage of the lysosomes, resulting in the accumulation of lysosomal lipids. An increased number of dark bodies is frequently observed in the epithelial cells of Reissner’s membrane in atoxyl intoxicated guinea pigs. However, it is difficult to establish their true identity without histochemical studies. The mesothelial cells adjacent to the scala vestibuli frequently exhibited protrusions of the cell membrane facing the perilymph. However, this only occurred when the total amount of atoxyl was very large and the specimen was investigated more than 24 hours after beginning the treatment. The cell organelles of the mesothelial cells took part in the degeneration in the same way as those in the epithelial cells but never showed such severe damage in the same specimen. The dark epithelial cells appear more distorted than the light cells, but the degeneration proceeds similarly, except that the vesiculation is minimal. No intermediate stages between the dark and light cells were observed. Acta OIolaryngol81
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422 M . Anniko
The changes in the position of Reissner’s membrane after atoxyl administration have earlier been described by Anniko & Wersall (1975). These changes indicate that strial damage causes a blockage of the endolymph circulation, resulting in an alteration in the position of the membrane. However, blockage of the transport activity in the Reissner’s membrane itself might also influence its position, provided passage of fluid and electrolytes by pinocytotic activity is taking place in the normal membrane. Atoxyl treatment thus results in damage to Reissner’s membrane which successively degenerates and loses its normal fluid transport function.
ZUSAMMENFASSUNG Uber die ototoxische Wirkung des arsenhaltigen Atoxyls auf die Feinstruktur der Stria vascularis wurde bereits berichtet (Anniko & Werslll. 1975). In dieser Studie werden die degenerativen Veranderungen der ReiRnerMembran nach Atoxyl-Applikation beschrieben. Schon 12 Stunden nach Atoxyl-Gabe kiinnen die ersten Degenerationszeichen der ReiRner-Membran feinstrukturell nachweisbar sein. Nach 24 Stunden ist die Schldigung gewohnlich sehr ausgeprigt. Epithelzellen und Mesothelzellen sind stark vakuolisiert, ihre Mitochondrien sind geschwollen und zeigen Bruche ihrer Cristae. im Cytoplasma sieht man Lipidgranula. Die Epithelzellen erscheinen starker geschldigt als die Mesothelzellen.
ACKNOWLEDGEMENT The author wishes to express his appreciation to Professor Jan Wersiill for his guidance. support and critical revision of the manuscript. The bkilful technical assistance of Mrs Marie-Louise SpAngberg and Mr Bengt Hedherg is gratefully acknowledged.
REFERENCES Anniko. M . & Werslll. J . 1975. Damage to the stria vascularis in the guinea pig by acute atoxyl intoxication. A m Otoltirwgol (Stockh)80. 167. von Bkkesy. C . 1953. Description of some mechanical properties of the organ of Corti. J Amrr.rr SOC A m 1.5. 770. Chou. J . T. 1963. Respiration of Keissner’s membrane of the guinea pig. J Ltrrynpol Oto/ 77. 374. Citron. L.. Exley, D. & Hsllpike. C . S. 1956. Formation. circulation and chemical properties of the litbyrinthine fluids. Rr ,bled Bull / 2 . 101.
Corti, A. 1851. Recherches sur I’organe de louie des mammiferes. Z Wiss Z o o / 3 , 109. Duvall, A. J. & Quick. A. C. 1%9. Tracers and endogenous debris in delineating cochlear barriers and pathways. Ann Otol Rhino/ L q w g o l 78, 1041. Duvall, A. J . & Rhodes, V. T. 1967. Reissner’s membrane-an ultrastructural study. Arch Oto/ary~i,qo/ 86. 37. Duvall. A. & Sutherland, C. R. 1970. The ultrastructure of the extrasensory cells in the cochlear duct. I n S y ~ n p o s i u mon Biochemicrrl Mechonisrns of Hecrriyc and Deafness, pp. 14%170. Ch. C. Thomas, Springfield, Ill. Duvall. A. J. & Tonndorf. J. 1962. Vital staining of the cochlea in guinea pigs. L ~ r y n g o s c o p e72, 892. Guild, S . R. 1927. The circulation of the endolymph. A m J Anrrt 39, 57. Hagiwara, S. 1963. Electron microscope study of the vestibular membrane (Reissner). Arch Histol Jrrp 24. 187. Hinojosa. R. 1971. Transport of ferritin acroks Reissner’s membrane. Acru Otoluvngol (Stockh). Suppl. 292. von Ilberg. C. 1968. Electronenmikroskopische Untersuchungen uber Diffusion und Resorption von T h e riumdioxid an der Meerschweinchenschnecke: 11. Reissnersche Mernbran. Arch O h r e t i Ntrseti KehlAopJheilhd 190. 426. lurato. S. 1967. Reissner’s membrane. In S u h r t i i c w .scopic~J’mrcturP c$ thr /mu Errr. Pergamon Press, Oxford. lurato, S . & Taidelli. G. 1%7. Struttura della membrana di Reissner. Boll Sot, / r t r / Biol Sper 4 3 , 1957. - 1971. The structure of the membrane of Reissner. II Internat. Symp. Gegenwartiger Stand der Cochlearforschung. Wiss Z Univ Hulle XX M, H. 1 , 105. Kaneko. Y.. Nakagawa. T. & Tanaka. K. 1970. Reissner’s membrane after kanamycin administration. Arch Otoluryngol92, 457. L.awrence. M.. Wolsk. D. & Litton. W. €3. 1961. Circulation of the inner ear fluids. Anti Otol Rhino/ l , o r y i i ,go/ 70. 753. Legouix. J . P. 1962. Electrophysiology. In Einfiihrrrlig i~ die Biocheriiic, dcs Hiiror.gr1n.r. Thieme, Stuttgart. Lundquist. P.-G. I%.(. The endolymphatic duct and s;ic in the guinea pig. A n electron microscopic and experimental investigation. Acfu Oroluryngol (Stockh), Suppl. 201. Misrahy, G.. Spradley; J . F.. Dzinovic. S . . Brooks. C . J . 1962. Effects of intense sound. hypoxia and kanamycin on the permeability of cochlear partitions. Anti Otol Rhino/ Ltrr,vtigo/ 70, 572. Naftalin. L. & Harrison. M . S. 1958. Circulation of labyrinthine fluids. J Lrrryyqol O t o / 71. 118. Nowak. R. 1974. Suhmikroskopixhe Veriindcrungen der Keissnerschen Membran nach Streptomycinapplikation. Actrr Oto/trryn,go/ (Stockh)77. 37. Plester. D., in discussion. Musebeck. K . 1963. Zuni Wirkungsmechanismus der Streptomycin-Vrrgiftung des Ohres. Arch 0hrc.n ,Ytr.sc,ri Kc4IAopjlicilAd 1x2. 583.
Downloaded by [University of New England] at 18:13 21 March 2016
Drimrrge t o Reissner’s membrane follottdng atoxyl intoxication Kauch. S. 1966. Membrane problems of the inner ear and their significance. J L ~ r y n g o Otol80. l 1144. Kauch. S . & Kostlin, A. 1958. Aspects chimiques de I’endolymphe et de la perilymphe. Prtrct OtorhinoItrryngol (Basel)20. 287. Kauch. S.. Kostlin, A , . Schneider. E.-A. & Schindler, K . 1963. Arguments for the permeability of Reissner’s membrane. Ltrryngoscope 78, 2. Kudert. H. 1969. Experimentelle Untersuchungen zur Resorption der Endolymphe im lnnenohr des Meerschweinchens: I. Lichtmikroskopische Untersuchungen nach lnjektion von Trypanblau in den Ductus cochlearis. Arch Ohren Nnsen KehlkopJ heilkd 193. 156. Schltzle. P. 1972. Personal communication. Ref. in Nowak. R. 1974. Submikroskopische Veranderungen der Reissnerschen Membran nach Streptomycinapplikation. Actn Otol~iryngol(Stockh) 77, 37. Smith, C. A , , Lowry, 0. H. & Wu. M. L., 1954. The electrolytes of the labyrinthine fluids. L(rryngoscope 64. 141.
423
Tasaki, I., Davis. H. & Eldredge. D. H. 1954. Exploration of cochlear potentials in guinea-pigs with a microelectrode. J Acoust Soc A m 26, 765. Tonndorf, J., Duvall, A . A. & Reneau, J. P. 1962. Permeability of intercochlear membrane to various stains. Ann Otol Rhino/ Lnryngol 73, 801. Watanuki, K. 1968. Some morphological observations of Reissner’s membrane. Actcr Otolaryngol (Stockh) 6 6 . 40. Watanuki. K., Hori, K., Kashiwazaki, H., Kaneko, Y., Kawamoto, K. & Katagiri, S. 1968. Some autoradiographic observations of the nucleic acid metabolism in the cells of Reissner’s membrane. Arch Klin Exp Ohren Nnsen Kehlkopfheilkd 192, 91,
M . Anniko, M.D. King Gustrrv V Research Institute S-10401 Stockholm 6 0 Snvden
Acto Orolaryngol8l
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Damage to reissner's membrane in the guinea-pig cochlea following acute atoxyl intoxication M. Anniko To cite this article: M. Anniko (1976) Damage to reissner's membrane in the guinea-pig cochlea following acute atoxyl intoxication, Acta Oto-Laryngologica, 81:5-6, 415-423, DOI: 10.3109/00016487609107495 To link to this article: http://dx.doi.org/10.3109/00016487609107495
Published online: 08 Jul 2009.
Submit your article to this journal
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 New England]
Date: 21 March 2016, At: 18:13
Acta Otolaryngol81: 415-423, 1976
DAMAGE TO REISSNER’S MEMBRANE IN THE GUINEA-PIG COCHLEA FOLLOWING ACUTE ATOXYL INTOXICATION M. Anniko Frorii the Deppcrrtnirtit of Otoltintigolog.v, Huddinge Sjuklius, the Krrrolinskri Institute. cind the King Gustin>V Resecirch Institute. the KurolitisXir Institute, Stockholm, Stt‘eden
Downloaded by [University of New England] at 18:13 21 March 2016
(Received August 28, 1975)
ing endolymph and perilymph is minimal. It may therefore play an important role in maintaining the difference in composition between the two fluids (Iurato, 1967). The relative proportions of electrolytes in the endolymph are quite different from those in the perilymph (Smith et al., 1954; Citron et al., 1956; Rauch & Kostlin, 1958). Two hypotheses have been advanced to explain how this difference in composition is maintained. According to the “longitudinal-flow’’ theory The ultrastructure of Reissner’s membrane (Corti, 1851; Guild, 1927; Lundquist, 1965) has been described in a number of publica- the endolymph is secreted in the stria vascutions (Hagiwara, 1963; Duvall & Rhodes, laris, runs along the cochlear duct and is re1967; Watanuki, 1968; Iurato & Taidelli, 1967; absorbed in the endolymphatic sac. This Duvall & Sutherland, 1970). The membrane is theory, which is supported by Tasaki et al. situated between the spiral limbus and the (1954) and Legouix (1962), also implies that vestibular crest of the spiral ligament; it is 2-3 Reissner’s membrane is practically impermenm thick, and consists of two layers. Towards able to sodium and potassium ions. On the the endolymphatic space there is a simple other hand, Naftalin & Harrison (1958), squamous epithelium. The surface facing the Lawrence et al. (1961) and Rauch et al. scala vestibuli consists of a layer of flat meso- (1963) support the “radial-flow’’ theory, i.e. thelial cells. A distinct basement membrane the hypothesis that the endolymph is formed is interposed between the two cell layers from peril ymph by passing through Reissner’s (Iurato & Taidelli, 1971). Reissner’s mem- membrane. According to this theory, the brane represents a portion of the membranous function of the stria vascularis is mainly ablabyrinth where the amount of tissue separat- sorptive. Compared to other tissues, the respiratory Supported by grants from the Karolinska Institute and rate of Reissner’s membrane is very high from the Swedish Medical Research Council (grant no. (Chou, 1963). Watanuki et al. (1968) showed 12X-720). Ahstroct. The ototoxic effects of the arsenic compound atoxyl has been earlier demonstrated by Anniko & Wersall (1975) in the stria vascularis of the guinea pig cochlea. The aim of the present investigation was to study the morphological degeneration at the ultrastructural level after damaging the Reissner’s membrane experimentally. I n acute atoxyl intoxication the first signs of degeneration appear after approximately I 2 hours. After 24 hours, the membrane may be markedly degenerated. Both the epithelial and mesothelial cells become vacuolized, the mitochondria lose their internal structure and become swollen, and lipid granules are found in the cytoplasm. Mesothelial cells are damaged to a lesser extent than the epithelial cells in the same specimen.
Acta Otolaryngol81
416 M . Anniko Table I . Administration of atoxyl to guinea pigs in the experimental group Amount injected on each occasion (mg/kg)
Animal no. ~
70 70
3 2
2 I /4
100
I
70 I40 I40
2
Total amount injected (mg/kg)
Interval between last injection and sacrifice (days)
Preyer reflex
~~~
20 52
53 54 55 63
Downloaded by [University of New England] at 18:13 21 March 2016
Number of injections
Injection period (days)
I /4
I 1
a higher turnover of nucleic acid (DNA) in the epithelial cells on the inner side of Reissner’s membrane than in the mesothelial cells on the outer side of it. Plester (1963) reported reduced protein synthesis in Reissner’s membrane after kanamycin administration. Misrahy et al. (1962) reported increased permeability after kanamycin treatment. Kaneko et al. (1970) suggest that after kanamycin administration Reissner’s membrane may lose its ability to maintain the normal chemical composition of the perilymph and endolymph. Tracers have been introduced into the scala vestibuli by many investigators (Tonndorf et al. 1962; Rudert, 1969; Duvall & Quick, 1969; von Ilberg, 1968; Rauch, 1966). There appears to be a selective passage of some dyes (methylene blue, toluidine blue and ferritin) through the membrane, while others (methyl blue, Janus green) do not pass through it (von Bekesy, 1953; Duvall & Tonndorf. 1962; Hinojosa, 1971). Nowak ( 1974) reported altered permeability of Reissner’s membrane following streptomycin administration. The aim of the present study was to investigate the effect on Reissner’s membrane of atoxyl (Pro Gen@ Sodium), an arsenic compound (sodium arsenilate) with known ototoxic properties (Anniko & Wersall. 1975). MATERIALS AND METHODS Nine healthy, young guinea pigs with a normal Preyer’s reflex, weighing around 25b.350 g were used for the experiment. Six animals Ac,fo O t i h r v n # i ~XI l
210 140
1
I /4
100
I
I40 I40 140
I /4
I I /2
were injected with sodium arsanilate (atoxyl). The control group consisted of 3 healthy, untreated guinea pigs. Each animal was injected subcutaneously with a 2 % solution of atoxyl in sterile water over a certain period of time. The amount of atoxyl injected on each occasion varied between 70 and 140 mg per kg body weight. The total dose ranged from 100-2 10 mg per kg body weight. The period of administration varied between 6 hours and 2 days and the survival time before decapitation between 6 hours and I day after the last injection (Table I). The specimens were treated according to standard methods for light and electron microscopy of osmium tetroxide-fixed specimens (Anniko & Wersall, 1975). RESULTS Structural alterations were observed as early as 12 hours after atoxyl administration. After 24-48 hours marked degeneration of Reissner’s membrane was observed. Administration of 70 mg of atoxyl per kg body weight twice within 48 hours and sacrificing the guinea pig 24 hours after the last injection resulted in very severe damage to the membrane in all coils, there being no apparent difference between the turns. Changes in all coils were observed when 70 mg of atoxyl per kg body weight was administered twice within 6 hours and the animal was sacrificed 12 hours after the first injection. The Reissner’s membrane in the basal coils in these animals was, however, less severely damaged
417
Downloaded by [University of New England] at 18:13 21 March 2016
Damage to Reissner’s membrane following atoxyl intoxication
Fig. I . The position of Reissner’s membrane is changed during the degeneration and it becomes depressed over
Fig. 2. Electron micrograph (EM). Degenerating endothelial ( E ) cell with massive vacuolization.
the organ of Corti as the damage proceeds. Light microscopy. x200. Acto Otoluryngol81
M.Anniko
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418
Fig. 3. (A) EM. Endothelial ( E )and mesothelial ( M ) cells
with mitochondria, showing fragmentation of their internal structure (arrows). (B) EM. Accumulations of electrondense material in the cytoplasm (mitochondria?) of endothelial ( E ) and mesothelial ( M ) cells (arrows). (C) EM.
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Differentiation of the degenerative pattern of the two cell types in Reissner’s membrane; the electron-dense appearance of the endothelial ( E ) cell cytoplasm can be seen. (D) EM.Lipoid-like accumulations in the cytoplasm of an endothelial ( E )cell.
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Accumulations of very electron-dense material in the cytoplasm of a “dark” endothelial ( E ) cell. Acta Ocolaryngol81
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than the rest of the cochlea. Animals which received 100 mg of atoxyl per kg body weight 24 hours before sacrifice showed only minimal degeneration of the Reissner’s membrane restricted to the apical coils. The same changes were observed when 140 mg of atoxyl per kg body weight was administered and the animals were sacrificed after the same time interval. The labyrinth from animals sacrificed 12 hours after such treatment showed no morphological changes. The position of Reissner’s membrane alters depending on the degree of intoxication. In some animals with early degeneration of the membrane the whole membrane was seen to bulge out towards the scala vestibuli (hydrops labyrinthine). In severe atoxyl intoxication the membrane become depressed and lies over the tectorial membrane and the Hensen cells but seldom touches the stria vascularis (Fig. I). All the cells within Reissner’s membrane were affected, the most severe degeneration being regularly found in the apical coils. Lesions occur both in the insertion of the membrane at the stria vascularis and in the area of the insertion at the spiral limbus. The squamous epithelial cells The squamous epithelium cells, which normally contains a moderate number of vesicles and pinocytotic vesicles, showed different degrees of vacuolization which was so widespread in some specimens that almost the whole cell was filled with vacuoles and the degenerating cell organelles are pressed towards the cell periphery (Fig. 2). The vacuolization becomes most widespread in the area around the nucleous. However, vacuoles of considerable size may also be observed in the region of the cell junction between two squamous epithelium cells. The normal cell appearance, with microvilli, tubules of the endoplasmic reticula, a small Golgi apparatus, a few microtubules and filaments and pinocytotic vesicles, is lost rather early during the degeneration process. Acta Otolaryngol81
Reductions in the numbers of microvilli and pinocytotic vesicles are the first signs of damage. The mitochondria swell and the cristae mitochondriales become fragmented (Fig. 3A). In severely damaged cells the mitochondria contain varying amounts of electrondense, osmiophilic material and this may even fill the mitochondrion completely (Fig. 3 B). Lipoid-like granules as large as 1&50 mitochondria are observed in the cytoplasm at all stages of degeneration (Fig. 3D). Some severely damaged squamous epithelium cells throughout the cytoplasm of the cell have an electron dense appearance (Fig. 3C). The cells are distorted but they still take part in the lining of Reissner’s membrane separating the two fluid compartments from each other (Fig. 4 A, B). The free surface of the epithelial cell layer towards the endolymph is uneven. However, free ribosomes, damaged mitochondria, fat granules and also accumulations of even more electron-dense material are also observed in these cells. The vacuolization is poor. The cell nucleous is resistant to degeneration and is the last cell component to disintegrate. It becomes involved in the final stage of damage by fragmentation of the chromatin and rupture of the nuclear membrane. The mesothelial cells The degeneration of the mesothelial cells follows a similar course to that of the epithelial cells. The cells become vacuolized, very few pinocytotic vesicles are observed and the internal structure of the mitochondria is fragmented but the mitochondria have fewer accumulations of electron dense material than those in the epithelial cells. Few fat granules are seen and they are not of the same size as those in the epithelial cells. Mesothelial cells with an electron-dense appearance similar to that of the cytoplasm of the whole cell have not been found. The mesothelial cells are always less degenerated than the squamous epithelial cells in the same specimen. Mesothelial cells adjacent to the scala vesti-
buli frequently contained rather large vacuoles on the cell membrane facing the perilymph. The degeneration of Reissner’s membrane starts in the apical part and it must undergo rather severe changes before its position changes. The electron-dense squamous epithelium cells were only found in animals where the Reissner’s membrane had changed from its normal position.
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DISCUSSION The present investigation reveals that atoxyl has a damaging effect on Reissner’s membrane in the guinea-pig. I n the initial phase of degeneration, no pathological changes are observed in the light microscope, while the electron microscope shows morphological alterations. Kaneko et al. (1970) reported that after kanamycin administration the epithelial cells in Reissner’s membrane showed vacuolization and condensation of cytoplasmic granules. They classified the cells into two types according to their cytoplasmic appearancelight and dark cells. The light cells showed vacuolization of the cytoplasm and little change in most of the cell organelles. On the other hand, the density of mitochondria and cell granules in the dark cells was increased. Both cell types showed decreased numbers of pinocytotic vesicles. The present results demonstrate the existence of some cells with comparatively dense cytoplasm. However, although the cell organelles in these “dark” cells are more electron dense than normal they take part in the degenerative process. It has been reported from electron microscopic studies that the epithelial cells of Reissner’s membrane contain many vesicles which may be involved in micropinocytosis. A well developed Golgi complex is found in the cytoplasm and many microvilli are observed on the endolymphatic surface (Hagiwara, 1%3; Iurato, 1967; Duvall & Modes, 1967). These findings suggest a high degree of metabolic activity.
In guinea-pigs made deaf by the administration of kanamycin, micropinocytotic vesicles were very rarely seen in the epithelial cells of Reissner’s membrane. Therefore, it is unlikely that active transport by the pinocytotic system is functional in these cells (Kaneko et al.. 1970). The two pathways from the scala vestibuli to the scala media through Reissner’s membrane via the intercellular junction and via pinocytosis have been demonstrated by Duvall & Quick (1969) and Duvall & Sutherland (1970). Misrahy et al. (1962) reported increased permeability after kanamycin administration, hypoxia and acoustic overstimulation. Nowak ( 1974) showed vacuolization. increased formation of lysosomes, lipid granules and an increased number of free ribosomes after dihydrostreptomycin administration. Fragmentation of the zonula occludents and desmosomes was also reported. Schatzle (1972) proposed that the lipid granules were formed by damage of the lysosomes, resulting in the accumulation of lysosomal lipids. An increased number of dark bodies is frequently observed in the epithelial cells of Reissner’s membrane in atoxyl intoxicated guinea pigs. However, it is difficult to establish their true identity without histochemical studies. The mesothelial cells adjacent to the scala vestibuli frequently exhibited protrusions of the cell membrane facing the perilymph. However, this only occurred when the total amount of atoxyl was very large and the specimen was investigated more than 24 hours after beginning the treatment. The cell organelles of the mesothelial cells took part in the degeneration in the same way as those in the epithelial cells but never showed such severe damage in the same specimen. The dark epithelial cells appear more distorted than the light cells, but the degeneration proceeds similarly, except that the vesiculation is minimal. No intermediate stages between the dark and light cells were observed. Acta OIolaryngol81
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The changes in the position of Reissner’s membrane after atoxyl administration have earlier been described by Anniko & Wersall (1975). These changes indicate that strial damage causes a blockage of the endolymph circulation, resulting in an alteration in the position of the membrane. However, blockage of the transport activity in the Reissner’s membrane itself might also influence its position, provided passage of fluid and electrolytes by pinocytotic activity is taking place in the normal membrane. Atoxyl treatment thus results in damage to Reissner’s membrane which successively degenerates and loses its normal fluid transport function.
ZUSAMMENFASSUNG Uber die ototoxische Wirkung des arsenhaltigen Atoxyls auf die Feinstruktur der Stria vascularis wurde bereits berichtet (Anniko & Werslll. 1975). In dieser Studie werden die degenerativen Veranderungen der ReiRnerMembran nach Atoxyl-Applikation beschrieben. Schon 12 Stunden nach Atoxyl-Gabe kiinnen die ersten Degenerationszeichen der ReiRner-Membran feinstrukturell nachweisbar sein. Nach 24 Stunden ist die Schldigung gewohnlich sehr ausgeprigt. Epithelzellen und Mesothelzellen sind stark vakuolisiert, ihre Mitochondrien sind geschwollen und zeigen Bruche ihrer Cristae. im Cytoplasma sieht man Lipidgranula. Die Epithelzellen erscheinen starker geschldigt als die Mesothelzellen.
ACKNOWLEDGEMENT The author wishes to express his appreciation to Professor Jan Wersiill for his guidance. support and critical revision of the manuscript. The bkilful technical assistance of Mrs Marie-Louise SpAngberg and Mr Bengt Hedherg is gratefully acknowledged.
REFERENCES Anniko. M . & Werslll. J . 1975. Damage to the stria vascularis in the guinea pig by acute atoxyl intoxication. A m Otoltirwgol (Stockh)80. 167. von Bkkesy. C . 1953. Description of some mechanical properties of the organ of Corti. J Amrr.rr SOC A m 1.5. 770. Chou. J . T. 1963. Respiration of Keissner’s membrane of the guinea pig. J Ltrrynpol Oto/ 77. 374. Citron. L.. Exley, D. & Hsllpike. C . S. 1956. Formation. circulation and chemical properties of the litbyrinthine fluids. Rr ,bled Bull / 2 . 101.
Corti, A. 1851. Recherches sur I’organe de louie des mammiferes. Z Wiss Z o o / 3 , 109. Duvall, A. J. & Quick. A. C. 1%9. Tracers and endogenous debris in delineating cochlear barriers and pathways. Ann Otol Rhino/ L q w g o l 78, 1041. Duvall, A. J . & Rhodes, V. T. 1967. Reissner’s membrane-an ultrastructural study. Arch Oto/ary~i,qo/ 86. 37. Duvall. A. & Sutherland, C. R. 1970. The ultrastructure of the extrasensory cells in the cochlear duct. I n S y ~ n p o s i u mon Biochemicrrl Mechonisrns of Hecrriyc and Deafness, pp. 14%170. Ch. C. Thomas, Springfield, Ill. Duvall. A. J. & Tonndorf. J. 1962. Vital staining of the cochlea in guinea pigs. L ~ r y n g o s c o p e72, 892. Guild, S . R. 1927. The circulation of the endolymph. A m J Anrrt 39, 57. Hagiwara, S. 1963. Electron microscope study of the vestibular membrane (Reissner). Arch Histol Jrrp 24. 187. Hinojosa. R. 1971. Transport of ferritin acroks Reissner’s membrane. Acru Otoluvngol (Stockh). Suppl. 292. von Ilberg. C. 1968. Electronenmikroskopische Untersuchungen uber Diffusion und Resorption von T h e riumdioxid an der Meerschweinchenschnecke: 11. Reissnersche Mernbran. Arch O h r e t i Ntrseti KehlAopJheilhd 190. 426. lurato. S. 1967. Reissner’s membrane. In S u h r t i i c w .scopic~J’mrcturP c$ thr /mu Errr. Pergamon Press, Oxford. lurato, S . & Taidelli. G. 1%7. Struttura della membrana di Reissner. Boll Sot, / r t r / Biol Sper 4 3 , 1957. - 1971. The structure of the membrane of Reissner. II Internat. Symp. Gegenwartiger Stand der Cochlearforschung. Wiss Z Univ Hulle XX M, H. 1 , 105. Kaneko. Y.. Nakagawa. T. & Tanaka. K. 1970. Reissner’s membrane after kanamycin administration. Arch Otoluryngol92, 457. L.awrence. M.. Wolsk. D. & Litton. W. €3. 1961. Circulation of the inner ear fluids. Anti Otol Rhino/ l , o r y i i ,go/ 70. 753. Legouix. J . P. 1962. Electrophysiology. In Einfiihrrrlig i~ die Biocheriiic, dcs Hiiror.gr1n.r. Thieme, Stuttgart. Lundquist. P.-G. I%.(. The endolymphatic duct and s;ic in the guinea pig. A n electron microscopic and experimental investigation. Acfu Oroluryngol (Stockh), Suppl. 201. Misrahy, G.. Spradley; J . F.. Dzinovic. S . . Brooks. C . J . 1962. Effects of intense sound. hypoxia and kanamycin on the permeability of cochlear partitions. Anti Otol Rhino/ Ltrr,vtigo/ 70, 572. Naftalin. L. & Harrison. M . S. 1958. Circulation of labyrinthine fluids. J Lrrryyqol O t o / 71. 118. Nowak. R. 1974. Suhmikroskopixhe Veriindcrungen der Keissnerschen Membran nach Streptomycinapplikation. Actrr Oto/trryn,go/ (Stockh)77. 37. Plester. D., in discussion. Musebeck. K . 1963. Zuni Wirkungsmechanismus der Streptomycin-Vrrgiftung des Ohres. Arch 0hrc.n ,Ytr.sc,ri Kc4IAopjlicilAd 1x2. 583.
Downloaded by [University of New England] at 18:13 21 March 2016
Drimrrge t o Reissner’s membrane follottdng atoxyl intoxication Kauch. S. 1966. Membrane problems of the inner ear and their significance. J L ~ r y n g o Otol80. l 1144. Kauch. S . & Kostlin, A. 1958. Aspects chimiques de I’endolymphe et de la perilymphe. Prtrct OtorhinoItrryngol (Basel)20. 287. Kauch. S.. Kostlin, A , . Schneider. E.-A. & Schindler, K . 1963. Arguments for the permeability of Reissner’s membrane. Ltrryngoscope 78, 2. Kudert. H. 1969. Experimentelle Untersuchungen zur Resorption der Endolymphe im lnnenohr des Meerschweinchens: I. Lichtmikroskopische Untersuchungen nach lnjektion von Trypanblau in den Ductus cochlearis. Arch Ohren Nnsen KehlkopJ heilkd 193. 156. Schltzle. P. 1972. Personal communication. Ref. in Nowak. R. 1974. Submikroskopische Veranderungen der Reissnerschen Membran nach Streptomycinapplikation. Actn Otol~iryngol(Stockh) 77, 37. Smith, C. A , , Lowry, 0. H. & Wu. M. L., 1954. The electrolytes of the labyrinthine fluids. L(rryngoscope 64. 141.
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Tasaki, I., Davis. H. & Eldredge. D. H. 1954. Exploration of cochlear potentials in guinea-pigs with a microelectrode. J Acoust Soc A m 26, 765. Tonndorf, J., Duvall, A . A. & Reneau, J. P. 1962. Permeability of intercochlear membrane to various stains. Ann Otol Rhino/ Lnryngol 73, 801. Watanuki, K. 1968. Some morphological observations of Reissner’s membrane. Actcr Otolaryngol (Stockh) 6 6 . 40. Watanuki. K., Hori, K., Kashiwazaki, H., Kaneko, Y., Kawamoto, K. & Katagiri, S. 1968. Some autoradiographic observations of the nucleic acid metabolism in the cells of Reissner’s membrane. Arch Klin Exp Ohren Nnsen Kehlkopfheilkd 192, 91,
M . Anniko, M.D. King Gustrrv V Research Institute S-10401 Stockholm 6 0 Snvden
Acto Orolaryngol8l
