- Email: [email protected]
Sequential changes in anti-GAL-1 staining of the rat organ of Corti following amikacin exposure
Brain Research 822 Ž1999. 43–51
Research report
Sequential changes in anti-GAL-1 staining of the rat organ of Corti following amikacin exposure M. Visitacion ´ Bartolome´ a , Philippe Vago b,c , Pablo Gil-Loyzaga a, ) , Gullienne Humbert b , Raymonde Joubert-Caron d , Remy ´ Pujol b , Marc Lenoir b a
Centro CultiÕos Celulares (CAI-UCM) and Departamento de Cirugıa ´ II (ORL), Facultad Medicina, UniÕersidad Complutense, Madrid, Spain b INSERM U. 254, UniÕersite´ Montpellier I, Faculte´ de Medecine, Montpellier, France ´ c Histologie-Embryologie-Cytogenetique, UniÕersite´ Montpellier I, Faculte´ de Medecine, Montpellier, France ´ ´ ´ d Laboratoire de Biochimie et Technologie des Proteines, UniÕersite´ Paris Nord, Bobigny, France ´ Accepted 15 December 1998
Abstract Hair cell loss and a non-functional epithelial reorganization appeared in the organ of Corti after acoustic or toxic damage. Moreover, in the drug damaged organ of Corti, transient atypical cells were recently described with characteristics of both immature hair cells andror non-sensory epithelial cells. The phenotype of these atypical cells has been now investigated by using the galectine 1 ŽGAL-1. antibody. In the normal organ of Corti, this antibody recognizes all the epithelial cells except the sensory hair cells and their supporting cells. At PD 21, transient atypical cells were not stained by GAL-1 antibody, suggesting that they were originated from hair cells or their supporting cells. Later, the organ of Corti was substituted by an epithelial scare, GAL-1 stained. This study also emphasizes the particular resistance of the cochlear apex to degeneration after antibiotic intoxication. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Transdifferentiation; b-galactoside; Lectin; Ototoxicity; Cochlea; Rat
1. Introduction In the mammalian organ of Corti, the inner ŽIHCs. and the outer ŽOHCs. hair cells are very sensitive to acoustic trauma or to drug toxicity. Whereas lost hair cells are replaced by new hair cells in the damaged basilar papilla of birds w1,7,8,35x, the loss of IHCs and OHCs is considered to be irreversible in adult mammals. In these conditions, the supporting cells exhibited an important spatial reorganization in vivo w5,10,17,23,28,29,34x and in vitro w36x. Amikacin, a well-known ototoxic aminoglycoside antibiotic, produced extensive hair cell losses w21x in particular during the period of cochlear enhanced sensitivity to ototoxicity, i.e., from postnatal day 9 to 16 w27x. Recently, the presence of atypical cells has been observed in the cochleae of young rats treated with amikacin w9,18,19,37x.
) Corresponding author. Universidad Complutense, Apartado de Correos 60.075, 28080 Madrid, Spain. Fax: q34-91-3941-383; E-mail: [email protected]
These atypical cells, occupying the region of the missing OHCs, shared morphological and phenotypical characteristics with both immature hair cells and non-sensory epithelial cells: their apical pole was covered with a tuft of actin rich microvilli and their cell body contained cytokeratins w9x. As proliferative cells were absent from the damaged region, it was hypothesized that atypical cells arise through direct transformation of some of the non-sensory epithelial cells which reorganize during and after hair cell degeneration w9,37x. The presence of these atypical cells in the organ of Corti after amikacin administration could suggest an attempt at hair cell transdifferentiation w9,18,19,37x. In the present study, we were interested in Ži. providing new information on the sequential changes occurring in the organ of Corti after amikacin administration, and ii. clarifying some aspects of phenotype of the transient atypical cells. The use of specific cell markers was required to differentiate sensory and non-sensory cell derivatives w13– 16,32,33x. In this way, we have investigated the immunohistochemical expression of galectine-1 ŽGAL-1., a b-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 0 7 0 - 7
44
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
galactoside-binding endogenous lectin w4,6x, in the organ of Corti of amikacin treated rats. In the developing and adult normal organ of Corti, this lectin was shown to be
specifically expressed in non-sensory cells but the supporting cells, i.e., border, phalangeal, pillar, and Deiters’ cells w11,31x. A total absence of this lectin was observed in the
Fig. 1. Scanning Ža, and insert. and transmission Žb. electron microscopy in amikacin treated rats ŽPD 21.. Ža. Surface view of the organ of Corti in the apical region of the cochlear spiral. All the hair cells are missing. Note the presence of small tufts of microvilli Žarrowheads. in the area of preexisting OHCs ŽO.. ŽP s pillar cells; ISS s inner spiral sulcus; OSS s outer spiral sulcus.. Scale bar s 50 mm. Insert: tufts of microvilli at higher magnification. Scale bar s 0.5 mm. Žb. Transverse section of the damaged organ of Corti in the lower apical region of the cochlea. The tunnel of Corti ŽtC. is largely open and the pillar cells Žp. look well-preserved. Both the OHCs and the IHCs are absent. Deiters’ cells ŽD. expansions have invaded the region of pre-existing OHCs. Epithelial cells of the OSS occupy a large area of the epithelium and partly cover the Deiters’ cells. Note: the two epithelial cells Ž1 and 2. in the area of the missing IHC. Scale bar s 20 mm.
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
Corti’s organ sensory cells during development and in the adulthood w11,31x. Amikacin was injected to rats from postnatal day 9 to 16 days, i.e., during the period of cochlear enhanced-sensitivity to ototoxicity w21,27x. The cochleae were processed for scanning ŽSEM., transmission ŽTEM. electron microscopy, and for immunostaining. They were examined at 21, 28, 45 and 70 days, when the cochlea is considered to be both anatomically w20,22x and functionally w26x adultlike.
45
2. Material and methods The care and use of animals in this study were in strict accordance with the animal welfare guidelines of the Declaration of Helsinki. A total number of 28 Wistar rats were used in this study. Twenty-four animals received a daily subcutaneous injection of 500 mgrkg of amikacin ŽBristol. for 8 consecutive days between postnatal day ŽPD. 9 and PD 16, the day of birth being PD 0. Twenty-one of these treated rats
Fig. 2. Surface preparations of the organ of Corti in the apical region of the cochlea Žconfocal microscopy.. Ža,b. Non-treated rat ŽPD 21.. Ža. Rhodamine phalloidin. Note the strong labelling at the level of the cuticular plates and stereocilia of OHCs ŽO. and IHCs ŽI. and at the level of the junctions between the Deiters’ cells and the OHCs ŽISS s inner spiral sulcus; OSS s outer spiral sulcus.. The pillars of Corti Žp. are also well labelled. Žb. Anti-GAL-1 immunoreactivity. Only the cells located in the OSS are labelled. Žc,d. Amikacin treated rat ŽPD 21.. Žc. Rhodamine phalloidin. The cuticular plates and stereocilia of the hair cells are absent. In the region of the pre-existing OHCs, some atypical cells Žarrowheads. can be identified: they showed a central dot of labelling corresponding to the actin present within the tuff of microvilli. Žd. Anti-GAL-1 immunoreactivity. The labelling is restricted to the cells in the OSS. The atypical cells are not stained. Scale bars s 10 mm.
46
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
were used for histochemical investigations: three of them were used for surface preparations and eighteen rats for paraffin sections at either PD 21 Ž n s 9., 28 Ž n s 3., 45 Ž n s 3. and 70 Ž n s 3.. Three treated rats were sacrificed at PD 21 for morphological investigations using SEM and TEM. Four non-treated ŽPD 21. rats served as control animals. 2.1. Electron microscopy The cochleae, from PD 21, were perilymphatically perfused with a fixative solution containing 2.5% glutaraldehyde in 0.1 M phosphate buffer saline at pH 7.4, 0.1 M ŽPBS.. Three cochleae were prepared for SEM, and one cochlea for TEM. For SEM, the bony capsule was removed, later the stria vascularis and the tectorial membrane were dissected away. Samples were dehydrated in graded series of ethanol from 50 to 100%, then critical point-dried in CO 2 . They were coated with 10 nm goldpalladium and observed under a Hitachi S4000 microscope. For TEM, the cochleae were postfixed in 1% aqueous osmium tetroxide, dehydrated in ethanol Ž50% to 100%. and embedded in Spurr resin. Five series of about 10 transverse ultrathin sections each, were performed in the apical region of each cochlea. Sections, mounted either on formvar coated or 200 mesh grids and counterstained with uranyl acetate and lead citrate, were examined using a Hitachi 7100 microscope. 2.2. Histochemistry 2.2.1. Surface preparations The left cochleae of three treated and two non-treated rats Žall sacrificed at PD 21. were fixed with paraformaldehyde 4% in PBS, washed twice then, preincubated for 30 min in a 30% goat serum solution in PBS. Subsequently, the apical coils were dissected away and prepared as surface preparations. Then, these surface preparations were processed for a double stained with anti-GAL-1 and actin. They were incubated for 2 h in a 1r2000 dilution of rabbit anti-GAL-1 monoclonal antibody then, after washing, the samples were incubated for 2 h in a solution containing fluorescein conjugated avidin ŽVector. Ž1r100
47
dilution.. For actin distribution the surface preparations were incubated 2 h in a 1r30 dilution of rhodamine phalloidin ŽMolecular Probes. in PBS. Finally, the specimens were washed twice and mounted between slide and a glass coverslip. Surface preparations were observed under a MRC 1200 laser scanning confocal microscope ŽBioRad.. Optical sections of 1 mm thickness were recorder and noise was reduced by Kalman averaging of 5 to 10 consecutive scans. The FITC Ž520 nm. and rhodamine Ž620 nm. fluorescences of 3 to 15 optical sections spaced from 1 to 5 mm were superimposed by computer to represent a single plane. 2.2.2. Paraffin sections The left cochleae of the 18 treated rats and the two non-treated rats were fixed by perilymphatic perfusion in 2% acetic acid in 70% ethanol, postfixed in the same solution for 72 h, dehydrated and embedded in paraffin. Samples were then serially sectioned at 7 mm thickness from the modiolar plane. The sections were rinsed three times, for 5 min each, in PBS. Preincubation was carried out for 30 min in a PBS solution and 30% goat serum. The sections were then incubated overnight, at 48C, in a 1r2000 solution of anti-GAL-1 monoclonal antibody w11,31x. After three washes Ž5 min each. in PBS, the sections were incubated for 1 h in biotinylated goat anti-rabbit IgG ŽVectastain, Vector, Laboratories. 1r200 in PBS. Antigen–antibody immunoreaction was revealed by using the avidin–biotin-peroxidase method ŽVectastain, Vector. as previously described w3,12,24x. Negative controls were obtained with the same procedure described above but omitting the primary antibody.
3. Results 3.1. Scanning electron microscopy Throughout the three cochleae investigated of PD 21 treated rats, the surface of the organ of Corti was devoid of both IHC and OHC stereociliary bundles. In the apical coil, small tufts of densely packed microvilli were seen in the area where OHCs are normally present ŽFig. 1a.. At
Fig. 3. Paraffin sections of the organ of Corti Žlight microscopy.. Control animals ŽC.; Treated rats with amikacin ŽAK.; basal, medial and apical coil ŽBC, MC, AC.. Ža. Non-treated rat, PD 21, medial coil. A strong positive immunoreaction is present in cells of the ISS Žasterisk. and of the OSS Žstar.. The three OHCs ŽO. and the IHC ŽI. are not labelled, nor the pillar cells Žp. and the Deiters’ cells ŽD.. The tunnel of Corti ŽtC. is largely open. Žb–g. Treated rats ŽAK.. Žb. PD 21, medial cochlear coil. The hair cells are missing. The region of pre-existing OHCs is covered by immunoreactive cells Žprobably the Hensen’s cells. and unlabelled or weakly labelled Žprobably the Deiters’ cells and their apical expansions.. The pillar cells are unlabelled or are weakly labelled. The cells in the region of the IHCs are not stained at all. Žc. PD 28, apical coil. The pattern of immunoreaction is essentially the same as in Žb., except that some cells are positive in the region of the Deiters’ cells. Žd,e. PD 45. Žd. Basal coil. All the cells of the epithelium are strongly labelled. Note the absence of the tunnel of Corti. Že. Medial coil of the same cochlea as that shown in Žd.. The tunnel of Corti is still open but the region of the external pillar and a part of the region of the Deiters’ cells appear immunoreactive. Žf–h. PD 70. Žf,g. Respectively the basal and the medial coil of the same cochlea. The whole epithelium is composed of a single layer of strongly immunoreactive cells. Žh. Apical coil of the cochlea shown in Žf. and Žg.. The pattern is essentially the same as that seen at PD 21 Žb. and PD 28 Žc.. Scale bars s 50 mm.
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
48
high magnification ŽFig. 1a, detail., these tufts were similar to the immature stereociliary bundles of cochlear hair cells. The number of tufts ranged from 28 to 86 tufts. 3.2. Transmission electron microscopy Although the organ of Corti already lacked both OHCs and IHCs, the general morphology of the epithelium was retained ŽFig. 1b.. The tunnel of Corti was still open. The Deiters’ cells had expanded in the Nuel’s spaces and in the area of the missing OHCs. In addition, the Hensen’s cells had partially invaded this area on the outer side. On the IHC side, two ovoid shaped epithelial cells were present in the area of the missing sensory cell. 3.3. Histochemistry 3.3.1. Surface preparations In non-treated rats, the cuticular plates and the stereociliary bundles of the hair cells, the head of the pillar cells, and the adherent junctions between the OHCs and the Deiters’ cells were strongly labelled with rhodamine phalloidin ŽFig. 2a.. These structures were not labelled with the anti-GAL-1 antibody ŽFig. 2b.. In contrast, the Hensen’s and the Claudius’ cells were strongly labelled with the anti-GAL-1 monoclonal antibody, and their junctions were weakly labelled with rhodamine phalloidin. In treated rats from PD 21, the cuticular plates and the stereocilia of the hair cells were absent. The adherent junctions at the surface of the epithelium and the head of the pillar cells were stained with rhodamine phalloidin ŽFig. 2c.. In the region of the pre-existing OHCs, some cells Ž10 in one sample and 22 in the other one. showed an
apical dot of rhodamine phalloidin ŽFig. 2c, arrows., which probably corresponded to the tufts of microvilli observed at the surface of the atypical cells under SEM ŽFig. 1a.. These cells were not immunoreactivity for anti-GAL-1 antibody ŽFig. 2d.. External to this region, a large band of epithelial cells was strongly immunolabelled with antiGAL-1 antibody, they probably corresponded the Hensen’s and Claudius’ cells. 3.3.2. Paraffin sections In non-treated rats, the results were identical to those previously reported in normal adult rats w11,31x. A strong positive immunoreaction was seen on both sides of the organ of Corti, in the epithelial cells of the inner spiral sulcus ŽISS. and of the outer spiral sulcus ŽOSS.. In contrast, a total absence of immunoreactivity was observed in both types of sensory cells ŽIHCs and OHCs., their specific supporting cells, border cells, phalangeal cells, inner and outer pillar cells and Deiters’ cells ŽFig. 3a.. In treated rats from PD 21, the pattern of distribution of anti-GAL-1 immunostaining was identical at all studied cochlear coils. A strong immunoreaction was seen in the epithelial cells of both the OSS and the ISS ŽFig. 3b.. In the OSS, labelled cells extended on the outer part of the pre-existing OHCs region, similar to that observed in surface preparation ŽFig. 2d.. Unlabelled cells, partially covered by labelled cells, were also seen in this region, from the basilar membrane to the top of the organ of Corti. In the ISS, all the epithelial cells were labelled except for the cells located in the region of IHCs. The tunnel of Corti appeared open ŽFig. 3b, star.. The pattern of distribution of anti-GAL-1 immunostaining was essentially the same between treated rats from
Table 1 Summary of results obtained by immunocytochemical distribution of anti-GAL-1 in different types of cells of the organ of Corti in the control and treated rats Rats
Basal coil
Medial coil
Apical coil
Control rats
IHCŽy., OHCŽy., DCŽy., ECŽq., ACŽnp. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽnp. Fig. 3d, IHCŽnp., OHCŽnp., DCŽnp., ECŽq., ACŽnp. Fig. 3f, IHCŽnp., OHCŽnp., DCŽnp., ECŽq., ACŽnp.
Fig. 3a, IHCŽy., OHCŽy., DCŽy., ECŽq., ACŽnp. Fig. 3b, IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. Fig. 3e, IHCŽy., OHCŽnp., DCŽnp., ECŽq., ACŽnp. Fig. 3g, IHCŽnp., OHCŽnp., DCŽnp., ECŽq., ACŽnp.
IHCŽy.,OHCŽy., DCŽy., ECŽq., ACŽnp. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. Fig. 3c, IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. Fig. 3h, IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy.
AK treated Ž21 PD. AK treated Ž28 PD. AK treated Ž45 PD. AK treated Ž70 PD.
AK: Amikacin. PD: Postnatal day. IHC: Inner hair cells. OHC: Outer hair cells. DC: Deiters’ cells. EC: Epithelial cells. AC: Atypical cells. np: Not present Žsee electron microscopy.. Žq.: Positive immunoreaction. Žy.: Negative immunoreaction.
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
PD 28 and PD 21. However, a weak immunoreaction was noticed in some cells located in the region of the missing OHCs, in particular at the cochlear apex region ŽFig. 3c.. An important change with respect to previous finding was observed in the basal coil of the cochleae from treated rats from PD 45. The tunnel of Corti collapsed, suggesting a receptorial degeneration, and all the remaining cells of the organ of Corti formed an epithelium, which showed a strong immunoreactivity ŽFig. 3d.. In the medial coil, only the epithelial cells of the IHCs area and inner pillar and head of external pillar cells showed an absence of immunoreactivity for anti-GAL-1 monoclonal antibody. The tunnel of Corti was still open ŽFig. 3e, star.. In the apical coil, the distribution of immunolabelling was similar to the apical coil of the cochlea observed in PD 28 treated rats. At PD 70 treated rats, in the basal ŽFig. 3f. and medial ŽFig. 3g. cochlear coils, the epithelium of the organ of Corti appeared as a single layer of strongly immunoreactive cells. At the apex ŽFig. 3h., the central region of the organ of Corti still showed an open tunnel ŽFig. 3h, star. and some immunoreactive cells in the area of the Deiters’ cells, as was observed in the previous stages ŽFig. 3c.. The immunohistochemical results obtained with antiGAL-1 monoclonal antibody in cochleae of control and treated rats were summarized in Table 1.
4. Discussion In the present experiment, animals were treated with 500 mgrkg of amikacin between 9 and 16 PD, i.e., during the period of cochlear enhanced-sensitivity to antibiotic w21,27x. In previous studies, such treatment was shown to produce extensive damage to OHCs and IHCs as early as PD 17. It also resulted in the transient presence, from PD 21 to PD 35, of atypical cells in the area of the missing OHCs in the apical coil of the cochlea w9,18,19x. The atypical cells were covered with round tufts of microvilli, similar to nascent stereociliary bundles of immature cochlear hair cells w20x. The number of atypical cells per cochlea varied from a few cells to several hundred w9x. The present results are consistent with these previous findings. They show a complete loss of both types of hair cells from PD 21, at least, to PD 70, the atypical cells were recognized at PD 21, in the region of the missing OHCs. The tufts of microvilli of the atypical cells were observed using SEM and identified under confocal microscopy using rhodamine phalloidin. The organ of Corti damaged by drugs administration w10,23,28x or noise exposed w29x, a scarring process rapidly occurs which protects the epithelium from further injury. These processes, involving the spatial reorganization of non-sensory epithelial cells, has been well described in the region of missing OHCs w9x. As illustrated by the present ultrastructural findings, the Deiters’ cells remained and the
49
Hensen’s cells invaded the Nuel’s spaces and the area of the pre-existing OHCs. When the anti-GAL-1 was used, the unlabelled cells, in amikacin ŽPD 21 and PD 28. treated rats, probably corresponded to pillars and Deiters’ cells Žw11,31x, see also the present results.. The labelled cells were probably Hensen’s cells or another cell type of the outer spiral sulcus, such as Claudius’ or Boettcher’s cells, which are now extended to ward the place of the missing OHCs. In fact, all these types of epithelial cells were also strongly labelled in the normal cochlea Žw11,31x, and the present results in controls.. Altogether, these results agree with the notion that all the non-sensory epithelial cells participate to the scarring process in the damaged organ of Corti w23x. The IHCs are generally less sensitive to noxious influences than OHCs, less is known about the scarring process occurring in the inner spiral sulcus. In previous investigations in the developing rat cochlea w31x, it has been hypothesized that the b-galactoside lectin expressed at the level of the GAL-1-immunoreactive cells blocks an excessive or abnormal nerve fiber distribution to non-sensory area. Thus, in amikacin treated cochleae, the progressive colonization of the scarring epithelium by GAL-1 immunoreactive cells could be a limiting factor to the maintenance of nerve fibers. Indirectly, this could have negative influences on the regenerative potential of the organ of Corti, at least on the survival of the atypical cells. Further morphological and molecular experiments are need to investigate the status of innervation of the amikacin treated organ of Corti. From recent investigations in cochleae of rats treated with amikacin antibiotic w18,19x, it has been hypothesized that the missing IHCs are spatially replaced by their supporting cells, i.e., the border cell on the internal side and the phalangeal cell on the external side. At the final stage of the scarring, after drug administration, acoustic trauma or genetic disorders, the non-functional organ of Corti is composed of a single layer of unidentified cuboid epithelial cells Žamong other examples see Refs. w25,30x.. In the present study, this pattern was seen at PD 45 in the basal coil of the cochlea and extended to the medial coils at PD 70 according to the baso-apical kinetics of the drug ototoxicity Žamong other examples see Ref. w2x and Table 1.. All the cells composing this epithelium were strongly immunoreactive to the anti-GAL-1 antibody. These features probably reveal a dramatic epithelial reorganization involving either a loss of the hair cell supporting cells, without excluding the possibility of a change in their phenotype. At PD 70, some cells of the organ of Corti, at its very apical region, still retain a recognizable shape and showed both GAL-1 non-immunoreactive and immunoreactive cells. This emphasizes the resistance of the cochlear apex to antibiotic injury. An other interest of the current experiment, was to clarify the phenotype of the transient atypical cells which appear in the OHCs region soon after amikacin administra-
50
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
tion. Such as hair cells and their supporting cells in the normal cochlea w31x, the atypical cells in the treated cochleae were not GAL-1 immunoreactive. This result supports the assumptions that Ži. atypical cells represent an attempt at hair cell transdifferentiation and Žii. arise from the transdifferentiation of supporting cells, probably Deiters’ cells w9,18,19,37x. However, this does not preclude that some Hensen’s cells which migrate toward the area of ototoxic damage and change their GAL-1 pattern of immunostaining participate to the production of the atypical cells. Further morphological and molecular experiments are needed to clarify the phenotype and innervation of the atypical cells which appear in the OHCs region after amikacin administration.
Acknowledgements We would like to thank Teresa Rodrıguez-Benito for ´ histological assistance, and J.L. Pasquier for photographic work. We also thank M. Galliego for animal care. A part of the morphological investigations were performed in Centre Regional d’Imagerie Cellulaire de Montpellier. This ´ work has been supported by grants from FISss 95-1540, French-Spanish Integrated Action ŽHF 94-231. and Human Capital and Mobility ERBCHRXCT940659.
References w1x H.J. Adler, Y. Raphael, New hair cells arise from supporting cell conversion in the acoustically damaged chick inner ear, Neurosci. Lett. 205 Ž1996. 17–20. w2x J.M. Aran, J.P. Erre, A. Guilhaume, C. Aurousseau, The comparative ototoxicities of gentamicin, tobramycin and dibekacin in the guinea pig. A functional and morphological cochlear and vestibular study, Acta Otolaryngol. ŽStockholm. 390 Ž1982. Suppl. w3x M.V. Bartolome, A. Merchan´ M.A. Ibanez, ´˜ J.G. Lopez-Sanchez, ´ ´ ´ Perez, P. Gil-Loyzaga, Synaptophysin immunoreactivity in the ´ cochlear nuclei of mammals: a comparative study, J. Otorhinolaryngol. Rel. Speciality ŽORL. 55 Ž1993. 317–321. w4x D. Bladier, J.P. Le Caer, R. Joubert, M. Caron, J. Rossier, bgalactoside soluble lectin from human brain. Complete aminoacid sequence, Neurochem. Int. 18 Ž1991. 275–281. w5x B.A. Bohne, Healing of the noise damaged inner ear, in: S.K. Hirsh, D.H. Eldredge, I.J. Hirsh, S.R. Silverman ŽEds.., Hearing and Davis: Essays Honoring Hallowell Davis, Washington Univ. Press, Saint Louis, MO, 1976, pp. 85–96. w6x M. Caron, D. Bladier, R. Joubert, Soluble galactoside-binding vertebrate lectins. A protein family with common properties, Int. J. Biochem. 22 Ž1990. 1379–1385. w7x J.T. Corwin, D.A. Cotanche, Regeneration of sensory cells after acoustic trauma, Science 240 Ž1988. 1772–1774. w8x D.A. Cotanche, Hair cell regeneration in the avian cochlea, Ann. Otol. Rhinol. Laryngol. 106 Ž1997. 9–15. w9x N. Daudet, P. Vago, C. Rippol, G. Humbert, R. Pujol, M. Lenoir, Characterization of atypical cells in the juvenile rat organ of Corti after aminoglycoside ototoxicity, J. Comp. Neurol. 401 Ž1998. 145– 162.
w10x A. Forge, Outer hair cell loss and supporting cell expansion following chronic gentamicin treatment, Hear. Res. 19 Ž1985. 171–182. w11x P. Gil-Loyzaga, M. Remezal, Glycoconjugates of the sensory hair cells of the organ of Corti, in: P. Gil-Loyzaga ŽEd.., Histochemistry of Glycoconjugates of the Auditory Receptor. Functional Implications, Gustav Fischer Verlag Sttutgart, Germany, 1997, pp. 44–78. w12x P. Gil-Loyzaga, M.V. Bartolome, ´ M.A. Vicente-Torres, Serotonergic innervation of the organ of corti of the cat cochlea, NeuroReport 8 Ž1997. 3519–3522. w13x P. Gil-Loyzaga, M. Remezal, R. Mollicone, A. Ibanez, ´˜ R. Oriol, H and B blood-group antigen expression in cochlear hair cells is modulated by thyroxine, Cell Tissue Res. 276 Ž1994. 239–243. w14x P. Gil-Loyzaga, M. Remezal, R. Oriol, Neuronal influence on B and H human blood-group antigen expression in rat cochlear cultures, Cell Tissue Res. 269 Ž1992. 13–20. w15x P. Gil-Loyzaga, R. Pujol, R. Mollicone, A. Dalix, R. Oriol, Appearance of B and H blood group antigens in the developing cochlear hair cells, Cell Tissue Res. 257 Ž1989. 17–21. w16x P. Gil-Loyzaga, W. Brownell, Wheat germ agglutinin and Helix pomatia agglutinin lectin binding on cochlear hair cells, Hear. Res. 34 Ž1988. 149–156. w17x J.E., Hawkins, Drug Ototoxicity, in: W.D. Keidel, W.D. Neff ŽEds.., Handbook of Sensory Physiology, Vol. 3, 1976, p. 707. w18x M. Lenoir, P. Vago, Does the organ of Corti attempt to differentiate new hair cells after antibiotic intoxication in rat pups?, Int. J. Dev. Neurosci. 15 Ž1997. 487–495. w19x M. Lenoir, P. Vago, Morphological indications of hair cell neodifferentiation in the organ of Corti of amikacin treated rat pups, C.R. Acad. Sci. Paris, Sciences de la VierLife Sciences 319 Ž1996. 269–276. w20x M. Lenoir, J.L. Puel, R. Pujol, Stereocilia and tectorial membrane development in the rat cochlea. A SEM study, Anat. Embryol. 175 Ž1987. 477–487. w21x M. Lenoir, M. Marot, A. Uziel, Comparative ototoxicity of four aminoglycoside antibiotics during development. A functional and morphological study in the rat, Acta Otolaryngol. ŽStockholm. 405 Ž1983. Suppl. w22x M. Lenoir, A. Shnerson, R. Pujol, Cochlear receptor development in the rat with emphasis on synaptogenesis, Anat. Embryol. 160 Ž1980. 253–262. w23x E.V. Leonova, Y. Raphael, Organization of cell junctions and cytoskeleton in the reticular lamina in normal and ototoxically damaged organ of Corti, Hear. Res. 113 Ž1997. 14–28. w24x A. Merchan, M. Eybalin, F.J. ´ P. Gil-Loyzaga, J.G. Lopez-Sanchez, ´ ´ Valderrama, Ontogeny of GABA in efferent fibers to the rat cochlea, Dev. Brain Res. 76 Ž1993. 33–41. w25x J.B. Nadol, Histopathology of human aminoglycoside ototoxicity, in: S.E., Lerner, G.J. Matz, J.E. Hawkins ŽEds.., Aminoglycoside Ototoxicity, Little, Brown and Company, Boston, 1981, pp. 409–434. w26x J.L. Puel, A. Uziel, Correlative development of cochlear action potential sensitivity, latency, and frequency selectivity, Dev. Brain Res. 37 Ž1987. 179–188. w27x R. Pujol, Period of sensitivity to antibiotic treatment in the developing cochlea, Acta Otolaryngol. ŽStockholm. 429 Ž1986. 29–33, Suppl. w28x Y. Raphael, R.A. Altschuler, Scar formation after drug-induced ¨ cochlear insult, Hear. Res. 51 Ž1991. 173–184. w29x Y. Raphael, R.A. Altschuler, Reorganization of cytoskeletal and ¨ junctional proteins during cochlear hair cell degeneration, Mot. Cytoskel. 18 Ž1991. 215–227. w30x M. Rebillard, R. Pujol, G. Rebillard, Variability of the hereditary deafness in the white cat: II. Histology, Hear. Res. 5 Ž1981. 189–200. w31x M. Remezal, P. Gil-Loyzaga, R. Mollicone, R. Joubert-Caron, R. Oriol, Histochemical localization of a b-galactoside-binding lectin and its binding-sites in developing and adult rat cochlea, Dev. Brain Res. 73 Ž1993. 1–6. w32x M. Remezal, P. Gil-Loyzaga, R. Mollicone, R. Oriol, Immunocyto-
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51 chemical approach to the structure of human blood groups B and H glycocongugate antigens in the three days old rat cochlea, Cell Tissue Res. 274 Ž1993. 21–26. w33x M. Remezal, P. Gil-Loyzaga, R. Oriol, Ultrastructural localization of H human-blood group immunoreactivity in rat cochlear hair cells, J. Otorhinolaryngol. Rel. Speciality ŽORL. 55 Ž1993. 205–207. w34x R. Romand, S. Chardin, S. Le Calvez, The spontaneous appearance of hair cell-like cells in the mammalian cochlea following aminoglycoside ototoxicity, NeuroReport 8 Ž1996. 133–137.
51
w35x B.M. Ryals, E.W. Rubel, Hair cell regeneration after acoustic trauma, Science 240 Ž1988. 1774–1776. w36x H.M. Sobkowicz, B.K. August, M. Slapnick, Epithelial repair following mechanical injury of the developing organ of Corti in culture: an electron microscopic and autoradiographic study, Exp. Neurol. 115 Ž1992. 44–49. w37x P. Vago, G. Humbert, M. Lenoir, Amikacin intoxication induces apoptosis and cell proliferation in rat organ of Corti, NeuroReport 9 Ž1998. 431–436.
Research report
Sequential changes in anti-GAL-1 staining of the rat organ of Corti following amikacin exposure M. Visitacion ´ Bartolome´ a , Philippe Vago b,c , Pablo Gil-Loyzaga a, ) , Gullienne Humbert b , Raymonde Joubert-Caron d , Remy ´ Pujol b , Marc Lenoir b a
Centro CultiÕos Celulares (CAI-UCM) and Departamento de Cirugıa ´ II (ORL), Facultad Medicina, UniÕersidad Complutense, Madrid, Spain b INSERM U. 254, UniÕersite´ Montpellier I, Faculte´ de Medecine, Montpellier, France ´ c Histologie-Embryologie-Cytogenetique, UniÕersite´ Montpellier I, Faculte´ de Medecine, Montpellier, France ´ ´ ´ d Laboratoire de Biochimie et Technologie des Proteines, UniÕersite´ Paris Nord, Bobigny, France ´ Accepted 15 December 1998
Abstract Hair cell loss and a non-functional epithelial reorganization appeared in the organ of Corti after acoustic or toxic damage. Moreover, in the drug damaged organ of Corti, transient atypical cells were recently described with characteristics of both immature hair cells andror non-sensory epithelial cells. The phenotype of these atypical cells has been now investigated by using the galectine 1 ŽGAL-1. antibody. In the normal organ of Corti, this antibody recognizes all the epithelial cells except the sensory hair cells and their supporting cells. At PD 21, transient atypical cells were not stained by GAL-1 antibody, suggesting that they were originated from hair cells or their supporting cells. Later, the organ of Corti was substituted by an epithelial scare, GAL-1 stained. This study also emphasizes the particular resistance of the cochlear apex to degeneration after antibiotic intoxication. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Transdifferentiation; b-galactoside; Lectin; Ototoxicity; Cochlea; Rat
1. Introduction In the mammalian organ of Corti, the inner ŽIHCs. and the outer ŽOHCs. hair cells are very sensitive to acoustic trauma or to drug toxicity. Whereas lost hair cells are replaced by new hair cells in the damaged basilar papilla of birds w1,7,8,35x, the loss of IHCs and OHCs is considered to be irreversible in adult mammals. In these conditions, the supporting cells exhibited an important spatial reorganization in vivo w5,10,17,23,28,29,34x and in vitro w36x. Amikacin, a well-known ototoxic aminoglycoside antibiotic, produced extensive hair cell losses w21x in particular during the period of cochlear enhanced sensitivity to ototoxicity, i.e., from postnatal day 9 to 16 w27x. Recently, the presence of atypical cells has been observed in the cochleae of young rats treated with amikacin w9,18,19,37x.
) Corresponding author. Universidad Complutense, Apartado de Correos 60.075, 28080 Madrid, Spain. Fax: q34-91-3941-383; E-mail: [email protected]
These atypical cells, occupying the region of the missing OHCs, shared morphological and phenotypical characteristics with both immature hair cells and non-sensory epithelial cells: their apical pole was covered with a tuft of actin rich microvilli and their cell body contained cytokeratins w9x. As proliferative cells were absent from the damaged region, it was hypothesized that atypical cells arise through direct transformation of some of the non-sensory epithelial cells which reorganize during and after hair cell degeneration w9,37x. The presence of these atypical cells in the organ of Corti after amikacin administration could suggest an attempt at hair cell transdifferentiation w9,18,19,37x. In the present study, we were interested in Ži. providing new information on the sequential changes occurring in the organ of Corti after amikacin administration, and ii. clarifying some aspects of phenotype of the transient atypical cells. The use of specific cell markers was required to differentiate sensory and non-sensory cell derivatives w13– 16,32,33x. In this way, we have investigated the immunohistochemical expression of galectine-1 ŽGAL-1., a b-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 0 7 0 - 7
44
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
galactoside-binding endogenous lectin w4,6x, in the organ of Corti of amikacin treated rats. In the developing and adult normal organ of Corti, this lectin was shown to be
specifically expressed in non-sensory cells but the supporting cells, i.e., border, phalangeal, pillar, and Deiters’ cells w11,31x. A total absence of this lectin was observed in the
Fig. 1. Scanning Ža, and insert. and transmission Žb. electron microscopy in amikacin treated rats ŽPD 21.. Ža. Surface view of the organ of Corti in the apical region of the cochlear spiral. All the hair cells are missing. Note the presence of small tufts of microvilli Žarrowheads. in the area of preexisting OHCs ŽO.. ŽP s pillar cells; ISS s inner spiral sulcus; OSS s outer spiral sulcus.. Scale bar s 50 mm. Insert: tufts of microvilli at higher magnification. Scale bar s 0.5 mm. Žb. Transverse section of the damaged organ of Corti in the lower apical region of the cochlea. The tunnel of Corti ŽtC. is largely open and the pillar cells Žp. look well-preserved. Both the OHCs and the IHCs are absent. Deiters’ cells ŽD. expansions have invaded the region of pre-existing OHCs. Epithelial cells of the OSS occupy a large area of the epithelium and partly cover the Deiters’ cells. Note: the two epithelial cells Ž1 and 2. in the area of the missing IHC. Scale bar s 20 mm.
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
Corti’s organ sensory cells during development and in the adulthood w11,31x. Amikacin was injected to rats from postnatal day 9 to 16 days, i.e., during the period of cochlear enhanced-sensitivity to ototoxicity w21,27x. The cochleae were processed for scanning ŽSEM., transmission ŽTEM. electron microscopy, and for immunostaining. They were examined at 21, 28, 45 and 70 days, when the cochlea is considered to be both anatomically w20,22x and functionally w26x adultlike.
45
2. Material and methods The care and use of animals in this study were in strict accordance with the animal welfare guidelines of the Declaration of Helsinki. A total number of 28 Wistar rats were used in this study. Twenty-four animals received a daily subcutaneous injection of 500 mgrkg of amikacin ŽBristol. for 8 consecutive days between postnatal day ŽPD. 9 and PD 16, the day of birth being PD 0. Twenty-one of these treated rats
Fig. 2. Surface preparations of the organ of Corti in the apical region of the cochlea Žconfocal microscopy.. Ža,b. Non-treated rat ŽPD 21.. Ža. Rhodamine phalloidin. Note the strong labelling at the level of the cuticular plates and stereocilia of OHCs ŽO. and IHCs ŽI. and at the level of the junctions between the Deiters’ cells and the OHCs ŽISS s inner spiral sulcus; OSS s outer spiral sulcus.. The pillars of Corti Žp. are also well labelled. Žb. Anti-GAL-1 immunoreactivity. Only the cells located in the OSS are labelled. Žc,d. Amikacin treated rat ŽPD 21.. Žc. Rhodamine phalloidin. The cuticular plates and stereocilia of the hair cells are absent. In the region of the pre-existing OHCs, some atypical cells Žarrowheads. can be identified: they showed a central dot of labelling corresponding to the actin present within the tuff of microvilli. Žd. Anti-GAL-1 immunoreactivity. The labelling is restricted to the cells in the OSS. The atypical cells are not stained. Scale bars s 10 mm.
46
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
were used for histochemical investigations: three of them were used for surface preparations and eighteen rats for paraffin sections at either PD 21 Ž n s 9., 28 Ž n s 3., 45 Ž n s 3. and 70 Ž n s 3.. Three treated rats were sacrificed at PD 21 for morphological investigations using SEM and TEM. Four non-treated ŽPD 21. rats served as control animals. 2.1. Electron microscopy The cochleae, from PD 21, were perilymphatically perfused with a fixative solution containing 2.5% glutaraldehyde in 0.1 M phosphate buffer saline at pH 7.4, 0.1 M ŽPBS.. Three cochleae were prepared for SEM, and one cochlea for TEM. For SEM, the bony capsule was removed, later the stria vascularis and the tectorial membrane were dissected away. Samples were dehydrated in graded series of ethanol from 50 to 100%, then critical point-dried in CO 2 . They were coated with 10 nm goldpalladium and observed under a Hitachi S4000 microscope. For TEM, the cochleae were postfixed in 1% aqueous osmium tetroxide, dehydrated in ethanol Ž50% to 100%. and embedded in Spurr resin. Five series of about 10 transverse ultrathin sections each, were performed in the apical region of each cochlea. Sections, mounted either on formvar coated or 200 mesh grids and counterstained with uranyl acetate and lead citrate, were examined using a Hitachi 7100 microscope. 2.2. Histochemistry 2.2.1. Surface preparations The left cochleae of three treated and two non-treated rats Žall sacrificed at PD 21. were fixed with paraformaldehyde 4% in PBS, washed twice then, preincubated for 30 min in a 30% goat serum solution in PBS. Subsequently, the apical coils were dissected away and prepared as surface preparations. Then, these surface preparations were processed for a double stained with anti-GAL-1 and actin. They were incubated for 2 h in a 1r2000 dilution of rabbit anti-GAL-1 monoclonal antibody then, after washing, the samples were incubated for 2 h in a solution containing fluorescein conjugated avidin ŽVector. Ž1r100
47
dilution.. For actin distribution the surface preparations were incubated 2 h in a 1r30 dilution of rhodamine phalloidin ŽMolecular Probes. in PBS. Finally, the specimens were washed twice and mounted between slide and a glass coverslip. Surface preparations were observed under a MRC 1200 laser scanning confocal microscope ŽBioRad.. Optical sections of 1 mm thickness were recorder and noise was reduced by Kalman averaging of 5 to 10 consecutive scans. The FITC Ž520 nm. and rhodamine Ž620 nm. fluorescences of 3 to 15 optical sections spaced from 1 to 5 mm were superimposed by computer to represent a single plane. 2.2.2. Paraffin sections The left cochleae of the 18 treated rats and the two non-treated rats were fixed by perilymphatic perfusion in 2% acetic acid in 70% ethanol, postfixed in the same solution for 72 h, dehydrated and embedded in paraffin. Samples were then serially sectioned at 7 mm thickness from the modiolar plane. The sections were rinsed three times, for 5 min each, in PBS. Preincubation was carried out for 30 min in a PBS solution and 30% goat serum. The sections were then incubated overnight, at 48C, in a 1r2000 solution of anti-GAL-1 monoclonal antibody w11,31x. After three washes Ž5 min each. in PBS, the sections were incubated for 1 h in biotinylated goat anti-rabbit IgG ŽVectastain, Vector, Laboratories. 1r200 in PBS. Antigen–antibody immunoreaction was revealed by using the avidin–biotin-peroxidase method ŽVectastain, Vector. as previously described w3,12,24x. Negative controls were obtained with the same procedure described above but omitting the primary antibody.
3. Results 3.1. Scanning electron microscopy Throughout the three cochleae investigated of PD 21 treated rats, the surface of the organ of Corti was devoid of both IHC and OHC stereociliary bundles. In the apical coil, small tufts of densely packed microvilli were seen in the area where OHCs are normally present ŽFig. 1a.. At
Fig. 3. Paraffin sections of the organ of Corti Žlight microscopy.. Control animals ŽC.; Treated rats with amikacin ŽAK.; basal, medial and apical coil ŽBC, MC, AC.. Ža. Non-treated rat, PD 21, medial coil. A strong positive immunoreaction is present in cells of the ISS Žasterisk. and of the OSS Žstar.. The three OHCs ŽO. and the IHC ŽI. are not labelled, nor the pillar cells Žp. and the Deiters’ cells ŽD.. The tunnel of Corti ŽtC. is largely open. Žb–g. Treated rats ŽAK.. Žb. PD 21, medial cochlear coil. The hair cells are missing. The region of pre-existing OHCs is covered by immunoreactive cells Žprobably the Hensen’s cells. and unlabelled or weakly labelled Žprobably the Deiters’ cells and their apical expansions.. The pillar cells are unlabelled or are weakly labelled. The cells in the region of the IHCs are not stained at all. Žc. PD 28, apical coil. The pattern of immunoreaction is essentially the same as in Žb., except that some cells are positive in the region of the Deiters’ cells. Žd,e. PD 45. Žd. Basal coil. All the cells of the epithelium are strongly labelled. Note the absence of the tunnel of Corti. Že. Medial coil of the same cochlea as that shown in Žd.. The tunnel of Corti is still open but the region of the external pillar and a part of the region of the Deiters’ cells appear immunoreactive. Žf–h. PD 70. Žf,g. Respectively the basal and the medial coil of the same cochlea. The whole epithelium is composed of a single layer of strongly immunoreactive cells. Žh. Apical coil of the cochlea shown in Žf. and Žg.. The pattern is essentially the same as that seen at PD 21 Žb. and PD 28 Žc.. Scale bars s 50 mm.
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
48
high magnification ŽFig. 1a, detail., these tufts were similar to the immature stereociliary bundles of cochlear hair cells. The number of tufts ranged from 28 to 86 tufts. 3.2. Transmission electron microscopy Although the organ of Corti already lacked both OHCs and IHCs, the general morphology of the epithelium was retained ŽFig. 1b.. The tunnel of Corti was still open. The Deiters’ cells had expanded in the Nuel’s spaces and in the area of the missing OHCs. In addition, the Hensen’s cells had partially invaded this area on the outer side. On the IHC side, two ovoid shaped epithelial cells were present in the area of the missing sensory cell. 3.3. Histochemistry 3.3.1. Surface preparations In non-treated rats, the cuticular plates and the stereociliary bundles of the hair cells, the head of the pillar cells, and the adherent junctions between the OHCs and the Deiters’ cells were strongly labelled with rhodamine phalloidin ŽFig. 2a.. These structures were not labelled with the anti-GAL-1 antibody ŽFig. 2b.. In contrast, the Hensen’s and the Claudius’ cells were strongly labelled with the anti-GAL-1 monoclonal antibody, and their junctions were weakly labelled with rhodamine phalloidin. In treated rats from PD 21, the cuticular plates and the stereocilia of the hair cells were absent. The adherent junctions at the surface of the epithelium and the head of the pillar cells were stained with rhodamine phalloidin ŽFig. 2c.. In the region of the pre-existing OHCs, some cells Ž10 in one sample and 22 in the other one. showed an
apical dot of rhodamine phalloidin ŽFig. 2c, arrows., which probably corresponded to the tufts of microvilli observed at the surface of the atypical cells under SEM ŽFig. 1a.. These cells were not immunoreactivity for anti-GAL-1 antibody ŽFig. 2d.. External to this region, a large band of epithelial cells was strongly immunolabelled with antiGAL-1 antibody, they probably corresponded the Hensen’s and Claudius’ cells. 3.3.2. Paraffin sections In non-treated rats, the results were identical to those previously reported in normal adult rats w11,31x. A strong positive immunoreaction was seen on both sides of the organ of Corti, in the epithelial cells of the inner spiral sulcus ŽISS. and of the outer spiral sulcus ŽOSS.. In contrast, a total absence of immunoreactivity was observed in both types of sensory cells ŽIHCs and OHCs., their specific supporting cells, border cells, phalangeal cells, inner and outer pillar cells and Deiters’ cells ŽFig. 3a.. In treated rats from PD 21, the pattern of distribution of anti-GAL-1 immunostaining was identical at all studied cochlear coils. A strong immunoreaction was seen in the epithelial cells of both the OSS and the ISS ŽFig. 3b.. In the OSS, labelled cells extended on the outer part of the pre-existing OHCs region, similar to that observed in surface preparation ŽFig. 2d.. Unlabelled cells, partially covered by labelled cells, were also seen in this region, from the basilar membrane to the top of the organ of Corti. In the ISS, all the epithelial cells were labelled except for the cells located in the region of IHCs. The tunnel of Corti appeared open ŽFig. 3b, star.. The pattern of distribution of anti-GAL-1 immunostaining was essentially the same between treated rats from
Table 1 Summary of results obtained by immunocytochemical distribution of anti-GAL-1 in different types of cells of the organ of Corti in the control and treated rats Rats
Basal coil
Medial coil
Apical coil
Control rats
IHCŽy., OHCŽy., DCŽy., ECŽq., ACŽnp. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽnp. Fig. 3d, IHCŽnp., OHCŽnp., DCŽnp., ECŽq., ACŽnp. Fig. 3f, IHCŽnp., OHCŽnp., DCŽnp., ECŽq., ACŽnp.
Fig. 3a, IHCŽy., OHCŽy., DCŽy., ECŽq., ACŽnp. Fig. 3b, IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. Fig. 3e, IHCŽy., OHCŽnp., DCŽnp., ECŽq., ACŽnp. Fig. 3g, IHCŽnp., OHCŽnp., DCŽnp., ECŽq., ACŽnp.
IHCŽy.,OHCŽy., DCŽy., ECŽq., ACŽnp. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. Fig. 3c, IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy. Fig. 3h, IHCŽy., OHCŽnp., DCŽy., ECŽq., ACŽy.
AK treated Ž21 PD. AK treated Ž28 PD. AK treated Ž45 PD. AK treated Ž70 PD.
AK: Amikacin. PD: Postnatal day. IHC: Inner hair cells. OHC: Outer hair cells. DC: Deiters’ cells. EC: Epithelial cells. AC: Atypical cells. np: Not present Žsee electron microscopy.. Žq.: Positive immunoreaction. Žy.: Negative immunoreaction.
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
PD 28 and PD 21. However, a weak immunoreaction was noticed in some cells located in the region of the missing OHCs, in particular at the cochlear apex region ŽFig. 3c.. An important change with respect to previous finding was observed in the basal coil of the cochleae from treated rats from PD 45. The tunnel of Corti collapsed, suggesting a receptorial degeneration, and all the remaining cells of the organ of Corti formed an epithelium, which showed a strong immunoreactivity ŽFig. 3d.. In the medial coil, only the epithelial cells of the IHCs area and inner pillar and head of external pillar cells showed an absence of immunoreactivity for anti-GAL-1 monoclonal antibody. The tunnel of Corti was still open ŽFig. 3e, star.. In the apical coil, the distribution of immunolabelling was similar to the apical coil of the cochlea observed in PD 28 treated rats. At PD 70 treated rats, in the basal ŽFig. 3f. and medial ŽFig. 3g. cochlear coils, the epithelium of the organ of Corti appeared as a single layer of strongly immunoreactive cells. At the apex ŽFig. 3h., the central region of the organ of Corti still showed an open tunnel ŽFig. 3h, star. and some immunoreactive cells in the area of the Deiters’ cells, as was observed in the previous stages ŽFig. 3c.. The immunohistochemical results obtained with antiGAL-1 monoclonal antibody in cochleae of control and treated rats were summarized in Table 1.
4. Discussion In the present experiment, animals were treated with 500 mgrkg of amikacin between 9 and 16 PD, i.e., during the period of cochlear enhanced-sensitivity to antibiotic w21,27x. In previous studies, such treatment was shown to produce extensive damage to OHCs and IHCs as early as PD 17. It also resulted in the transient presence, from PD 21 to PD 35, of atypical cells in the area of the missing OHCs in the apical coil of the cochlea w9,18,19x. The atypical cells were covered with round tufts of microvilli, similar to nascent stereociliary bundles of immature cochlear hair cells w20x. The number of atypical cells per cochlea varied from a few cells to several hundred w9x. The present results are consistent with these previous findings. They show a complete loss of both types of hair cells from PD 21, at least, to PD 70, the atypical cells were recognized at PD 21, in the region of the missing OHCs. The tufts of microvilli of the atypical cells were observed using SEM and identified under confocal microscopy using rhodamine phalloidin. The organ of Corti damaged by drugs administration w10,23,28x or noise exposed w29x, a scarring process rapidly occurs which protects the epithelium from further injury. These processes, involving the spatial reorganization of non-sensory epithelial cells, has been well described in the region of missing OHCs w9x. As illustrated by the present ultrastructural findings, the Deiters’ cells remained and the
49
Hensen’s cells invaded the Nuel’s spaces and the area of the pre-existing OHCs. When the anti-GAL-1 was used, the unlabelled cells, in amikacin ŽPD 21 and PD 28. treated rats, probably corresponded to pillars and Deiters’ cells Žw11,31x, see also the present results.. The labelled cells were probably Hensen’s cells or another cell type of the outer spiral sulcus, such as Claudius’ or Boettcher’s cells, which are now extended to ward the place of the missing OHCs. In fact, all these types of epithelial cells were also strongly labelled in the normal cochlea Žw11,31x, and the present results in controls.. Altogether, these results agree with the notion that all the non-sensory epithelial cells participate to the scarring process in the damaged organ of Corti w23x. The IHCs are generally less sensitive to noxious influences than OHCs, less is known about the scarring process occurring in the inner spiral sulcus. In previous investigations in the developing rat cochlea w31x, it has been hypothesized that the b-galactoside lectin expressed at the level of the GAL-1-immunoreactive cells blocks an excessive or abnormal nerve fiber distribution to non-sensory area. Thus, in amikacin treated cochleae, the progressive colonization of the scarring epithelium by GAL-1 immunoreactive cells could be a limiting factor to the maintenance of nerve fibers. Indirectly, this could have negative influences on the regenerative potential of the organ of Corti, at least on the survival of the atypical cells. Further morphological and molecular experiments are need to investigate the status of innervation of the amikacin treated organ of Corti. From recent investigations in cochleae of rats treated with amikacin antibiotic w18,19x, it has been hypothesized that the missing IHCs are spatially replaced by their supporting cells, i.e., the border cell on the internal side and the phalangeal cell on the external side. At the final stage of the scarring, after drug administration, acoustic trauma or genetic disorders, the non-functional organ of Corti is composed of a single layer of unidentified cuboid epithelial cells Žamong other examples see Refs. w25,30x.. In the present study, this pattern was seen at PD 45 in the basal coil of the cochlea and extended to the medial coils at PD 70 according to the baso-apical kinetics of the drug ototoxicity Žamong other examples see Ref. w2x and Table 1.. All the cells composing this epithelium were strongly immunoreactive to the anti-GAL-1 antibody. These features probably reveal a dramatic epithelial reorganization involving either a loss of the hair cell supporting cells, without excluding the possibility of a change in their phenotype. At PD 70, some cells of the organ of Corti, at its very apical region, still retain a recognizable shape and showed both GAL-1 non-immunoreactive and immunoreactive cells. This emphasizes the resistance of the cochlear apex to antibiotic injury. An other interest of the current experiment, was to clarify the phenotype of the transient atypical cells which appear in the OHCs region soon after amikacin administra-
50
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51
tion. Such as hair cells and their supporting cells in the normal cochlea w31x, the atypical cells in the treated cochleae were not GAL-1 immunoreactive. This result supports the assumptions that Ži. atypical cells represent an attempt at hair cell transdifferentiation and Žii. arise from the transdifferentiation of supporting cells, probably Deiters’ cells w9,18,19,37x. However, this does not preclude that some Hensen’s cells which migrate toward the area of ototoxic damage and change their GAL-1 pattern of immunostaining participate to the production of the atypical cells. Further morphological and molecular experiments are needed to clarify the phenotype and innervation of the atypical cells which appear in the OHCs region after amikacin administration.
Acknowledgements We would like to thank Teresa Rodrıguez-Benito for ´ histological assistance, and J.L. Pasquier for photographic work. We also thank M. Galliego for animal care. A part of the morphological investigations were performed in Centre Regional d’Imagerie Cellulaire de Montpellier. This ´ work has been supported by grants from FISss 95-1540, French-Spanish Integrated Action ŽHF 94-231. and Human Capital and Mobility ERBCHRXCT940659.
References w1x H.J. Adler, Y. Raphael, New hair cells arise from supporting cell conversion in the acoustically damaged chick inner ear, Neurosci. Lett. 205 Ž1996. 17–20. w2x J.M. Aran, J.P. Erre, A. Guilhaume, C. Aurousseau, The comparative ototoxicities of gentamicin, tobramycin and dibekacin in the guinea pig. A functional and morphological cochlear and vestibular study, Acta Otolaryngol. ŽStockholm. 390 Ž1982. Suppl. w3x M.V. Bartolome, A. Merchan´ M.A. Ibanez, ´˜ J.G. Lopez-Sanchez, ´ ´ ´ Perez, P. Gil-Loyzaga, Synaptophysin immunoreactivity in the ´ cochlear nuclei of mammals: a comparative study, J. Otorhinolaryngol. Rel. Speciality ŽORL. 55 Ž1993. 317–321. w4x D. Bladier, J.P. Le Caer, R. Joubert, M. Caron, J. Rossier, bgalactoside soluble lectin from human brain. Complete aminoacid sequence, Neurochem. Int. 18 Ž1991. 275–281. w5x B.A. Bohne, Healing of the noise damaged inner ear, in: S.K. Hirsh, D.H. Eldredge, I.J. Hirsh, S.R. Silverman ŽEds.., Hearing and Davis: Essays Honoring Hallowell Davis, Washington Univ. Press, Saint Louis, MO, 1976, pp. 85–96. w6x M. Caron, D. Bladier, R. Joubert, Soluble galactoside-binding vertebrate lectins. A protein family with common properties, Int. J. Biochem. 22 Ž1990. 1379–1385. w7x J.T. Corwin, D.A. Cotanche, Regeneration of sensory cells after acoustic trauma, Science 240 Ž1988. 1772–1774. w8x D.A. Cotanche, Hair cell regeneration in the avian cochlea, Ann. Otol. Rhinol. Laryngol. 106 Ž1997. 9–15. w9x N. Daudet, P. Vago, C. Rippol, G. Humbert, R. Pujol, M. Lenoir, Characterization of atypical cells in the juvenile rat organ of Corti after aminoglycoside ototoxicity, J. Comp. Neurol. 401 Ž1998. 145– 162.
w10x A. Forge, Outer hair cell loss and supporting cell expansion following chronic gentamicin treatment, Hear. Res. 19 Ž1985. 171–182. w11x P. Gil-Loyzaga, M. Remezal, Glycoconjugates of the sensory hair cells of the organ of Corti, in: P. Gil-Loyzaga ŽEd.., Histochemistry of Glycoconjugates of the Auditory Receptor. Functional Implications, Gustav Fischer Verlag Sttutgart, Germany, 1997, pp. 44–78. w12x P. Gil-Loyzaga, M.V. Bartolome, ´ M.A. Vicente-Torres, Serotonergic innervation of the organ of corti of the cat cochlea, NeuroReport 8 Ž1997. 3519–3522. w13x P. Gil-Loyzaga, M. Remezal, R. Mollicone, A. Ibanez, ´˜ R. Oriol, H and B blood-group antigen expression in cochlear hair cells is modulated by thyroxine, Cell Tissue Res. 276 Ž1994. 239–243. w14x P. Gil-Loyzaga, M. Remezal, R. Oriol, Neuronal influence on B and H human blood-group antigen expression in rat cochlear cultures, Cell Tissue Res. 269 Ž1992. 13–20. w15x P. Gil-Loyzaga, R. Pujol, R. Mollicone, A. Dalix, R. Oriol, Appearance of B and H blood group antigens in the developing cochlear hair cells, Cell Tissue Res. 257 Ž1989. 17–21. w16x P. Gil-Loyzaga, W. Brownell, Wheat germ agglutinin and Helix pomatia agglutinin lectin binding on cochlear hair cells, Hear. Res. 34 Ž1988. 149–156. w17x J.E., Hawkins, Drug Ototoxicity, in: W.D. Keidel, W.D. Neff ŽEds.., Handbook of Sensory Physiology, Vol. 3, 1976, p. 707. w18x M. Lenoir, P. Vago, Does the organ of Corti attempt to differentiate new hair cells after antibiotic intoxication in rat pups?, Int. J. Dev. Neurosci. 15 Ž1997. 487–495. w19x M. Lenoir, P. Vago, Morphological indications of hair cell neodifferentiation in the organ of Corti of amikacin treated rat pups, C.R. Acad. Sci. Paris, Sciences de la VierLife Sciences 319 Ž1996. 269–276. w20x M. Lenoir, J.L. Puel, R. Pujol, Stereocilia and tectorial membrane development in the rat cochlea. A SEM study, Anat. Embryol. 175 Ž1987. 477–487. w21x M. Lenoir, M. Marot, A. Uziel, Comparative ototoxicity of four aminoglycoside antibiotics during development. A functional and morphological study in the rat, Acta Otolaryngol. ŽStockholm. 405 Ž1983. Suppl. w22x M. Lenoir, A. Shnerson, R. Pujol, Cochlear receptor development in the rat with emphasis on synaptogenesis, Anat. Embryol. 160 Ž1980. 253–262. w23x E.V. Leonova, Y. Raphael, Organization of cell junctions and cytoskeleton in the reticular lamina in normal and ototoxically damaged organ of Corti, Hear. Res. 113 Ž1997. 14–28. w24x A. Merchan, M. Eybalin, F.J. ´ P. Gil-Loyzaga, J.G. Lopez-Sanchez, ´ ´ Valderrama, Ontogeny of GABA in efferent fibers to the rat cochlea, Dev. Brain Res. 76 Ž1993. 33–41. w25x J.B. Nadol, Histopathology of human aminoglycoside ototoxicity, in: S.E., Lerner, G.J. Matz, J.E. Hawkins ŽEds.., Aminoglycoside Ototoxicity, Little, Brown and Company, Boston, 1981, pp. 409–434. w26x J.L. Puel, A. Uziel, Correlative development of cochlear action potential sensitivity, latency, and frequency selectivity, Dev. Brain Res. 37 Ž1987. 179–188. w27x R. Pujol, Period of sensitivity to antibiotic treatment in the developing cochlea, Acta Otolaryngol. ŽStockholm. 429 Ž1986. 29–33, Suppl. w28x Y. Raphael, R.A. Altschuler, Scar formation after drug-induced ¨ cochlear insult, Hear. Res. 51 Ž1991. 173–184. w29x Y. Raphael, R.A. Altschuler, Reorganization of cytoskeletal and ¨ junctional proteins during cochlear hair cell degeneration, Mot. Cytoskel. 18 Ž1991. 215–227. w30x M. Rebillard, R. Pujol, G. Rebillard, Variability of the hereditary deafness in the white cat: II. Histology, Hear. Res. 5 Ž1981. 189–200. w31x M. Remezal, P. Gil-Loyzaga, R. Mollicone, R. Joubert-Caron, R. Oriol, Histochemical localization of a b-galactoside-binding lectin and its binding-sites in developing and adult rat cochlea, Dev. Brain Res. 73 Ž1993. 1–6. w32x M. Remezal, P. Gil-Loyzaga, R. Mollicone, R. Oriol, Immunocyto-
M.V. Bartolome´ et al.r Brain Research 822 (1999) 43–51 chemical approach to the structure of human blood groups B and H glycocongugate antigens in the three days old rat cochlea, Cell Tissue Res. 274 Ž1993. 21–26. w33x M. Remezal, P. Gil-Loyzaga, R. Oriol, Ultrastructural localization of H human-blood group immunoreactivity in rat cochlear hair cells, J. Otorhinolaryngol. Rel. Speciality ŽORL. 55 Ž1993. 205–207. w34x R. Romand, S. Chardin, S. Le Calvez, The spontaneous appearance of hair cell-like cells in the mammalian cochlea following aminoglycoside ototoxicity, NeuroReport 8 Ž1996. 133–137.
51
w35x B.M. Ryals, E.W. Rubel, Hair cell regeneration after acoustic trauma, Science 240 Ž1988. 1774–1776. w36x H.M. Sobkowicz, B.K. August, M. Slapnick, Epithelial repair following mechanical injury of the developing organ of Corti in culture: an electron microscopic and autoradiographic study, Exp. Neurol. 115 Ž1992. 44–49. w37x P. Vago, G. Humbert, M. Lenoir, Amikacin intoxication induces apoptosis and cell proliferation in rat organ of Corti, NeuroReport 9 Ž1998. 431–436.