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Synaptophysin immunohistochemistry in the human cochlea
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Hearing Research 185 (2003) 35^42 www.elsevier.com/locate/heares
Synaptophysin immunohistochemistry in the human cochlea Shaden Ali M. Khalifa a , Ulla Friberg a , Robert-Benjamin Illing b , Helge Rask-Andersen b
a;
a Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital, 751 85 Uppsala, Sweden Neurobiological Research Laboratory, Department of Otorhinolaryngology, Albert-Ludwigs-University Freiburg, 79106 Freiburg, Germany
Received 4 March 2003; accepted 23 July 2003
Abstract Light microscopy and immunohistochemical analyses of a freshly prepared human cochlea, removed at meningioma skull base surgery, were performed with particular emphasis on synaptophysin (SY) reactivity. Synaptophysin, a 38-kDa glycoprotein, is one of the most abundant integral membrane proteins of small presynaptic vesicles and is a useful marker for sites of synaptic transmission of the efferent olivocochlear system in the cochlea. Following fixation and decalcification, cryosections of 30 Wm were prepared. To introduce immunostaining, free-floating sections were exposed to monoclonal SY antibody. Positive SY immunostaining was solely restricted to the neural and sensory structures and did not include supporting cells of the organ of Corti. Dense reaction products were noted around the hair cells, especially at the basal portion of the inner and outer hair cells and their neural poles, as well as around the inner spiral bundle, tunnel spiral bundle, outer spiral bundle and upper tunnel crossing fibers. The majority of spiral ganglion cells stained positively. An intermingling network of thin unmyelinated nerve fibers stained densely, especially at the basal portions of the cochlea. The spiral limbus, inner and outer sulcus cells, basilar membrane, myelinated nerve fibers, spiral ligament and the stria vascularis were unstained. Human cochlea obtained during surgery offers excellent conditions for immunohistochemical analysis. In the basal cochlea in the organ of Corti, outer hair cell area, there may be alterations due to noise trauma from the drilling procedure. 5 2003 Elsevier B.V. All rights reserved. Key words: Cochlea; Organ of Corti ; Synaptophysin; Human; Noise trauma; Outer hair cell; Inner hair cell
1. Introduction Of the two types of hair cells found in the organ of Corti, inner hair cells (IHCs) and outer hair cells (OHCs), IHCs are the primary transducers of sensory information. The role of the OHCs is still under debate, but they seem to protect the IHCs from loud noise and may also modulate the sound signal. IHCs form chemical synapses with dendrites of type I spiral ganglion neurons (SGNs) that constitute 90^95% of the SGNs.
* Corresponding author. Tel.: +46 (18) 6115303; Fax: +46 (18) 6115360. E-mail address: [email protected] (H. Rask-Andersen).
OHCs synapse on the remaining type II SGNs (Spoendlin, 1979). Synaptophysin (SY), a 38-kDa glycoprotein, is one of the most abundant integral membrane proteins of small presynaptic vesicles and is also a major calcium-binding protein (Rehm et al., 1986). The protein is widely distributed at synapses throughout the nervous system, where it is believed to be involved in the exocytosis of stored neurotransmitter. SY-38, a monoclonal antibody directed against the C-terminus of SY, has proved to be a useful marker for sites of synaptic transmission of the e¡erent olivocochlear system innervating IHCs and OHCs, and has been used to map such transmission in various animals and in humans. Numerous animal studies (Sokolowski and Cunningham, 1996; Gil-Loyzaga and Pujol, 1988; Knipper et al., 1995; Simmons et al., 1996; Liberman et al., 1990; Heinrich et al., 1997; Counter et al., 1997), and a few
0378-5955 / 03 / $ ^ see front matter 5 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-5955(03)00228-4
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human studies (Anniko et al., 1989, 1995; Nadol et al., 1993; Nadol and Burgess, 1994; Rask-Andersen et al., 2000), have shown the presence of SY in e¡erent neural structures in the organ of Corti and spiral ganglion. SY immunoreactivity has been proved to be very sensitive to prolonged ¢xation, and of poor quality following formaldehyde and paraformaldehyde ¢xation. Knowledge of antigen decay due to postmortem artifacts is essential for the correct evaluation of immunoperoxidase studies of reduced immunoreactivity (i.e. a decreased number of SY-positive synapses). The present investigation is the ¢rst to analyze the human organ of Corti immunohistochemically in freshly obtained surgical tissue at varying levels from base to apex using serial cryosectioning of the cochlea. The aim of the study was to elucidate if the use of fresh human inner ear tissue for immuno-studies would give better results than postmortem ¢xed tissue. Another aim was to elucidate if surgically induced cochlear hyperstimulation (drilling) prior to ¢xation would a¡ect immunostaining properties by comparing the staining patterns at di¡erent regions of the cochlea. It is known that high noise levels generated during drilling of long duration may cause temporary threshold shifts (TTS) of up to 5^40 dB (Kylen and Arlinger, 1976; Kylen et al., 1977). Such changes may be partly associated with noise-induced morphological alterations which might a¡ect both a¡erent and e¡erent function and thereby immunostaining (Robertson, 1983).
2. Materials and methods 2.1. Patient data This study is based on the analysis of a human cochlea from a 41-year-old woman with a left-sided 3-cm cerebellopontine angle meningioma. Her preoperative hearing level assessed with four-frequency pure-tone average at 500, 1000, 2000, and 3000 Hz levels was normal (12.5 dB). The removal of cochlear tissue during translabyrinthine surgery for cerebellopontine angle meningioma for the purpose of morphological investigations was approved by the human subjects committee at the Uppsala University Hospital, Sweden. 2.2. Immunohistochemistry The patient su¡ered from a cerebellopontine meningioma which was surgically removed by a transcochlear/petrosal route. The inner ear was surgically removed by drilling and the complete cochlea was carefully drilled out and dissected in one piece. The duration of dissection and drilling to secure the cochlea was approximately 10 min. The bony cochlea was immediately transferred to ice-cold ¢xative containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 15% saturated picric acid in 0.1 M phosphate bu¡er at pH 7.4, where it remained for 72 h. Following ¢xation, decalci¢cation was done in two steps. The cochlea was incu-
Fig. 1. Low-power photomicrograph showing SY-positive spiral nerve structures in the osseous spiral lamina (OSL) in a fresh surgically obtained human cochlea (radial section, middle cochlear turn). Transversely cut immunopositive nerve bundles (arrowheads) are seen together with dense staining in the region of the organ of Corti (OC). SL: spiral limbus. Original magni¢cation U140.
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bated in 8% EDTA in phosphate bu¡er (0.1 M, pH 7.4) at 4‡C for 1 week, followed by another 2 weeks in 25% EDTA at 37‡C. This was found to have decalci¢ed the cochlear bone su⁄ciently to make 30-Wm frozen sections. To introduce immunostaining, free-£oating sections were exposed to three pre-incubation solutions : 45 min in 0.05% H2 O2 , 30 min in 1% milk powder, and ¢nally 30 min in blocking bu¡er containing 10% non-immune bovine serum and 0.05% Triton X-100 in phosphate bu¡er, all at room temperature. Subsequently, sections were exposed to monoclonal SY-38 antibody (Boehringer Mannheim, Germany) at a concentration of 0.14 Wg/ml. After an incubation time of 72 h at 4‡C, binding sites of the receptor antibodies were detected using the avidin^biotin technique (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine (0.05%) and ammonium nickel sulfate (0.3%). Finally, sections were mounted on gelatinized slides, dehydrated in a graded series of ethanol, and coverslipped in Entellan (Merck, Germany).
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2.3. Light microscopy (LM) The organ of Corti was sectioned separately from the lower basal, upper basal, lower middle, upper middle and apical regions. The di¡erent turns were analyzed using LM.
3. Results 3.1. Spiral nerve ¢bers Spiral ¢bers appeared in small bundles that were four to six in number. The myelinated ¢bers in the proximal osseous spiral lamina were unstained, while the ¢bers appearing at the transitional zone and peripheral to the habenula perforata showed strong SY immunoreaction (Fig. 1). 3.2. Organ of Corti The radial a¡erents and lateral e¡erents innervating
Fig. 2. Higher-power photomicrograph of the human organ of Corti demonstrated in Fig. 1. SY-positive nerve ¢bers can be seen at the level of the habenula perforata (arrowhead). Positive staining is also seen at the neural poles of IHCs and OHCs. Inner (IP) and outer pillar cells (OP) are unstained. SL: spiral limbus. ISC: inner sulcus cells. Original magni¢cation U490.
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Fig. 3. Adjacent section showing SY staining below and adjacent to the IHCs and OHCs (arrowheads). Some staining probably also occurs in the IHCs. The tunnel spiral bundle (TSB; mediating e¡erents to the OHC region) is positively stained. IPC: inner pillar cell. OPC: outer pillar cell. BM: basilar membrane. V: spiral vein. Original magni¢cation U1200.
the IHCs coursed together and stained strongly SY-positive (Figs. 1 and 2). It was not possible to distinguish between a¡erent and e¡erent ¢bers innervating the IHCs (Fig. 3). Innervation of the OHCs showed strong SY immunostaining of the medial e¡erents, represented by the upper tunnel crossing ¢bers and tunnel spiral bundle (Fig. 4). Upper tunnel crossing ¢bers and tunnel spiral bundle SY immunoreactivity sometimes had a ‘beaded’ or varicose appearance. SY immunoreactivity was equally strong in nerve endings below the OHCs and IHCs (Figs. 1^4). 3.3. Hair cells The hair cells also showed positive SY staining. The basal two thirds of the £ask-shaped bodies of the IHCs were stained with a gradual weakening towards the apex. The nucleus, the cytoplasm below the cuticular plate, and the stereocilia appeared unstained (Fig. 4). The rod-shaped OHCs stained more uniformly throughout the body of the cell, although the cuticular
plate and stereocilia remained unstained (Figs. 3 and 4). 3.4. Supporting cells Inner and outer pillar cells were unstained, as were Deiters’ cells (Fig. 4). 3.5. Spiral ganglion The major part of large spiral ganglion cells (type I) proved SY-positive, while a few showed only faint staining (Fig. 5). One or two appeared unstained. Small ganglion cells (type II) appeared to be SY-positive, although some heterogeneity in staining properties was noted. Myelinated nerve ¢bers were immunonegative. Unmyelinated ¢bers were strongly SY-positive and had a beaded or varicose appearance. These ¢bers intermingled with spiral ganglion cells and a close physical relationship was sometimes noted. The unmyelinated ¢bers merged into a larger bundle constituting the
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Fig. 4. LM of the human organ of Corti at the upper basal region of a fresh surgically obtained human cochlea. The tunnel spiral bundle (TSB) and upper tunnel crossing ¢bers (e¡) are SY-positive. Dense staining products occur below the OHCs and IHCs. Stereocilia and cuticular plate of the IHC can be seen (arrowhead). Some staining also occurs in the cytoplasm of the hair cells. OP: outer pillar cell. IP: inner pillar cell. BM: basilar membrane. V: spiral vessel. Original magni¢cation U700.
e¡erent intraganglionic spiral bundle, which was also strongly SY-positive (Fig. 5). 3.6. Radial topographic variations Radially, the strength of SY staining decreased somewhat from the ¢rst to the third row of the OHCs (Fig. 3). 3.7. Longitudinal topographic variations The SY staining pattern along the basal, middle and apical turns was not uniform in the various cell structures of the organ of Corti. The IHCs and nerve ¢ber structures around the IHCs stained rather equally from base to apex. The OHCs stained more weakly towards the apical region, as did the e¡erent nerve terminals and e¡erent nerve ¢ber structures. Towards the basal region, the OHCs were sometimes clustered into one entity, rendering it impossible to discern the individual OHCs. Towards the apex, the OHCs were seen to gain length and were separated into three or four distinguishable rows (Figs. 3 and 4).
4. Discussion The monoclonal antibody for SY has a⁄nity for identi¢ed presynaptic vesicle glycoproteins and has been used as an intracochlear marker for e¡erents of the olivocochlear bundle. Experimental animal studies have shown a reaction with anti-SY in the sensory cells of the avian cochlea (Sokolowski and Cunningham, 1996). In the developing rat and guinea pig, SY immunolabeling marked the varicosities of the olivocochlear e¡erent endings around IHCs and OHCs (Gil-Loyzaga and Pujol, 1988), and also monitored growth and synaptogenesis of e¡erents in postnatal development of the inner ear in the rat and hamster (Knipper et al., 1995; Simmons et al., 1996). In the cat, SY visualized e¡erent nerve endings at the IHCs and OHCs (Liberman et al., 1990). Strong SY immunoreactivity was present in the e¡erent nerve endings near the base of IHCs and OHCs as well as in the cytoplasm of OHCs in guinea pig studies (Heinrich et al., 1997). Another guinea pig study using quantitative analysis of SY immuno£uorescence demonstrated e¡erent nerve endings at the IHCs to be smaller, more numerous, and more densely packed
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Fig. 5. LM showing SY immunohistochemistry of the human spiral ganglion of the middle cochlear turn of a fresh surgically obtained human cochlea. Most ganglion cells show strong immunopositivity while a few show only faint staining (arrowheads). Myelinated nerve ¢bers are immunonegative while many unmyelinated ¢bers show positive reaction. The unmyelinated ¢bers merged into a larger bundle constituting the efferent intraganglionic spiral bundle (IGSB). These ¢bers are frequently physically related to the spiral ganglion cells. Original magni¢cation U390.
compared to OHC e¡erent nerve endings. This study also showed a longitudinal gradient with a higher percentage of e¡erent terminals innervating the IHCs and OHCs in the basal and mid segments than in the apical regions (Counter et al., 1997). Liberman et al. (1990) noted e¡erents to the OHCs to be most numerous at the basal cochlear turns between the 6- and 24-kHz region, whereas they found e¡erents to the IHCs to be uniformly distributed throughout the cochlea. Earlier studies on the human inner ear showed staining by SY in the apical region of both OHCs and IHCs as well as in adjacent nerve terminals in the fetal inner ear. Adult hair cells were shown to have immunoreactivity throughout the cytoplasm (Anniko et al., 1989). In more recent studies using neonatal and infant human temporal bones, SY was found at the base of the IHCs and OHCs, and in the inner spiral bundle, outer spiral bundle, tunnel spiral bundle and upper tunnel crossing ¢bers (Nadol et al., 1993). Anti-SY reactivity also showed decreasing immunoreactivity from base to apex and from the ¢rst to third OHC rows (Nadol et al., 1993). Using transmission electron microscopy, the same group (Nadol and Burgess, 1994) also showed
labeled e¡erent synapses on Deiters’ cells. Another study on the adult human inner ear showed SY expressed only in spiral ganglion cells, in contrast to the calcium-binding protein S-100 which occurred more generally in the labyrinth (Anniko et al., 1995). Spiral ganglion cells were mostly SY-positive, as were unmyelinated beaded nerve ¢bers and bundles associated with the ganglion cells in preparations of freshly obtained inner ear tissue (Rask-Andersen et al., 2000). The degree of antigen decay due to postmortem artifacts and ¢xation could account for the heterogeneous staining patterns of hair cells in di¡erent studies. Aspeci¢c background staining is known to increase postmortem and SY immunoreactivity and has been proved to be very sensitive to prolonged ¢xation (Sillevis Smitt et al., 1993). In the present study, the use of a fresh surgically obtained cochlea with a short ¢xation and decalci¢cation time minimized SY antigen decay. There was excellent preservation of antigenic sites throughout the cochlea. The SY-positive labeling of neuronal and sensory structures of the cochlea was in accordance with earlier studies on humans. However, at the light microscopic level it was not possible to make distinctions
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between stained and unstained nerve endings below hair cells, which would represent the e¡erent and a¡erent systems, respectively. Fine ¢bers known to represent the olivocochlear system, such as the inner spiral bundle, upper tunnel crossing ¢bers, and outer spiral ¢bers, were strongly SY-positive, indicating that they were efferent ¢bers which couple to the OHCs and synaptic a¡erent terminals of the IHCs. The ¢ndings in the present study regarding di¡erences in staining of IHCs and OHCs could represent nerve endings terminating at the supranuclear region of the OHCs as described by Liberman et al. (1990) and Nadol et al. (1993). The morphology and course of SY-positive nerve ¢bers indicate possible synaptic contacts with spiral ganglion cells. In the present study the IHC staining properties did not show signi¢cant variation longitudinally. This was not in accord with earlier studies by Counter et al. (1997), who showed more e¡erent terminals innervating the IHCs at the basal and middle segments of the cochlea using quantitative analysis of SY immuno£uorescence. The present results were more in accord with those of Liberman et al. (1990), who found e¡erents to the IHCs to be uniformly distributed throughout the cochlea. The OHCs stained more weakly towards the apical region and this was also in accord with ¢ndings of Liberman et al. (1990), who noted e¡erents to the OHCs to be most numerous at the basal cochlear turns between the 6- and 24-kHz region. Earlier transmission electron microscopy studies have revealed that synaptic contact sites are frequently encountered on the small cells (type II) and sometimes even on the large cells (type I) in the human spiral ganglion. Such synaptic contacts have been observed at sites where unmyelinated ¢bers from the e¡erent intraganglionic spiral bundle are in close association with the type II spiral ganglion cells. The positive staining of most of the small ganglion cells is in accordance with previous studies indicating close connections between e¡erents of the olivocochlear bundle and a¡erent neurons within the spiral ganglion suggesting that the OHC system may be under the in£uence of e¡erent innervation at the level of the spiral ganglion (Rask-Andersen et al., 2000). The noise levels reaching the cochlea during drilling of the mastoid process, as measured in human cadavers and temporal bones, showed that the ipsilateral cochlea was exposed to noise levels of about 100 dB (Kylen and Arlinger, 1976). Drilling on the ossicular chain generated vibratory stimulation equivalent to sound pressure levels on the eardrum higher than 130 dB (Helms, 1976). TTSs have been described after ear surgery for chronic otitis media and varied between 5 and 40 dB, with a positive correlation between magnitude of TTS and duration of noise exposure. The TTS was detected
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by means of pre- and post-exposure electrocochleograms. The frequencies primarily a¡ected were 4 and 8 kHz (Kylen et al., 1977). Bone drilling in the meningioma patient in this study was conducted for approximately 5 h, followed by 10 min of drilling directly on the cochlear otic capsule before ¢xation. Such energy levels could theoretically lead to structural noise damage projecting especially around the vulnerable 4-kHz area in the basal turn of the cochlea. To date, no study using anti-SY labeling of the organ of Corti has been performed immediately after noise trauma in experimental animals. One SY study in the guinea pig was performed after a 1-month recovery period. The weak SY activity at the lesion sites was speculated to be due to a reduction in number or size of terminal e¡erent endings or synaptic vesicles. Exhaustion of synaptic vesicle stores after exposure to continuous intense noise stimulation might alter e¡erent ending reactivity (Canlon et al., 1999). Morphological studies in the organ of Corti of experimental animals in general using light and electron microscopy have shown that OHCs are more vulnerable to acoustic trauma than IHCs, and particularly in the basal turn. Nerve endings are more resistant (Lim, 1976). In rats where one ear was exposed to 130 dB for 30 min, signi¢cantly more damage to the transductional apparatus was found at high-frequency regions as compared to low-frequency regions (Michler and Illing, 2002). In rabbits exposed to 100 dB for 2 h, examination in the acute stage, using electron microscopy, showed that the infranuclear region of OHCs had the most damage, and this was related to the degree of change seen in the e¡erent nerve endings (Omata et al., 1992). The e¡erent terminals showed dilated mitochondria and a decrease in number of small vesicles immediately after acoustic exposure (Omata and Schatzle, 1984). The acute changes, however, are mostly reported to be subtle. Chan et al. (1998) reported a reduction of sti¡ness and cell length of the OHCs in the acute post-exposure phase. Nordmann et al. (2000) observed a buckling of the pillar bodies in chinchillas after 24 h of exposure to 86 dB and noted that OHC stereocilia were not embedded in the tectorial membrane at the site of the noise-induced TTS. In the present study the morphology of the organ of Corti in basal turn appeared altered. It is not known whether the cause of these changes, occurring mainly in the OHC region, was a preparational artifact or if the changes re£ect functional modi¢cations due to acoustic hyperstimulation occurring prior to ¢xation. In that case the variances in staining properties may correspond to various frequency regions. The SY labeling in the basal turn, giving the impression of OHCs lumped together, could be in accordance with alterations noted earlier after acoustic trauma in animals,
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e.g. shortening of the OHCs as described by Chan et al. (1998) and buckling of pillar bodies (Nordmann et al., 2000). To draw conclusions from morphological changes in the experimental animal to the human organ of Corti can, however, be dubious and interindividual variations in susceptibility to noise trauma between humans make it hard to predict morphological changes. The study con¢rmed that fresh surgically obtained human cochlear tissue is well suited for immunohistochemical studies. One disadvantage could be that the specimens might be a¡ected by acoustic overload causing cellular and subcellular changes. It is unclear if acoustic stimulation of 100 dB for 5 h is su⁄cient to modify the synaptic and OHC architecture in the human inner ear and whether this may lead to the occurrence of structural damage before the ¢xation. The excellent preservation of antigenic sites could o¡er an opportunity to study how functional modi¢cations may a¡ect results of inner ear immunolabeling.
Acknowledgements The authors wish to thank Anders Kinnefors for the skillful surgical procedure involved in removing the cochlea, and Ingvor Forsberg for skillful technical assistance. The study was supported by grants from the Swedish Research Council Project 3908 and from Stiftelsen Tysta Skolan.
References Anniko, M., Arnold, W., Thornell, L.E., 1989. Localization of the integral membrane glycoprotein synaptophysin and the surface glycoprotein Egp-34 in the embryonic and adult human inner ear. ORL J. Otorhinolaryngol. Relat. Spec. 51, 221^228. Anniko, M., Arnold, W., Stigbrand, T., Stro«m, A., 1995. The human spiral ganglion. ORL J. Otorhinolaryngol. Relat. Spec. 57, 68^77. Canlon, B., Fransson, A., Viberg, A., 1999. Medial olivocochlear e¡erent terminals are protected by sound conditioning. Brain Res. 850, 253^260. Chan, E., Suneson, A., Ulfendahl, M., 1998. Acoustic trauma causes reversible sti¡ness changes in auditory sensory cells. Neuroscience 83, 961^968. Counter, S.A., Canlon, B., Borg, E., Aldskogius, H., 1997. Pattern of synaptophysin immunoreactivity in the e¡erent nerve terminals of the guinea pig cochlea. Neurosci. Lett. 222, 199^203. Gil-Loyzaga, P., Pujol, R., 1988. Synaptophysin in the developing cochlea. Int. J. Dev. Neurosci. 6, 155^160. Heinrich, U.R., Maurer, J., Backes, K., Mann, W., 1997. Immunoelectron-microscopic localization of synaptophysin in the organ of
Corti of the guinea pig. ORL J. Otorhinolaryngol. Relat. Spec. 59, 317^321. Helms, J., 1976. Acoustic trauma from the bone cutting burr. J. Laryngol. Otol. 90, 1143^1149. Knipper, M., Zimmermann, U., Rohbock, K., Kopschall, I., Zenner, H.P., 1995. Synaptophysin and GAP-43 proteins in e¡erent ¢bers of the inner ear during postnatal development. Dev. Brain Res. 89, 73^86. Kylen, P., Arlinger, S., 1976. Drill-generated noise levels in ear surgery. Acta Otolaryngol. (Stockh.) 82, 402^409. Kylen, P., Arlinger, S., Bergholtz, L.M., 1977. Peroperative temporary threshold shift in ear surgery. An electrocochleographic study. Acta Otolaryngol. (Stockh.) 84, 393^401. Liberman, M.C., Dodds, L.W., Pierce, S., 1990. A¡erent and e¡erent innervation of the cat cochlea: quantitative analysis with light and electron microscopy. J. Comp. Neurol. 301, 443^460. Lim, D.J., 1976. Ultrastructural cochlear changes following acoustic hyperstimulation and ototoxicity. Ann. Otol. Rhinol. Laryngol. 85, 740^751. Michler, S.A., Illing, R.B., 2002. Acoustic trauma induces re-emergence of the growth and plasticity associated protein GAP-43 in the rat auditory brainstem. J. Comp. Neurol. 451, 250^266. Nadol, J.B., Burgess, B.J., 1994. Supranuclear e¡erent synapses on outer hair cells and Deiters’ cells in the human organ of Corti. Hear. Res. 81, 49^56. Nadol, J.B., Cho, Y.B., Burgess, B.J., Adams, J.C., 1993. The localization of synaptophysin in the organ of Corti of the human as shown by immunoelectron microscopy. Acta Otolaryngol. (Stockh.) 113, 312^317. Nordmann, A.S., Bohne, B.A., Harding, G.W., 2000. Histopathological di¡erences between temporary and permanent threshold shift. Hear. Res. 139, 13^30. Omata, T., Omata, E., Wilhelms, H.J., Schatzle, W., 1992. Neural and infranuclear region changes in outer hair cells in acoustically exposed rabbits. Eur. Arch. Otorhinolaryngol. 249, 287^292. Omata, T., Schatzle, W., 1984. Electron microscopical studies on the e¡ect of lapsed time on the nerve endings of the outer hair cells in acoustically exposed rabbits. Arch. Otorhinolaryngol. 240, 175^ 183. Rask-Andersen, H., Tylstedt, S., Kinnefors, A., Illing, R.-B., 2000. Synapses on human spiral ganglion cells: a transmission electron microscopy and immunohistochemical study. Hear. Res. 141, 1^ 11. Rehm, H., Wiedenmann, B., Betz, H., 1986. Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane. EMBO J. 5, 535^541. Robertson, D., 1983. Functional signi¢cance of dendritic swelling after loud sounds in the guinea pig cochlea. Hear. Res. 9, 263^278. Sillevis Smitt, P.A., van der Loos, C., Vianney de Jong, J.M., Troost, D., 1993. Tissue ¢xation methods alter the immunohistochemical demonstrability of neuro¢lament proteins, synaptophysin, and glial ¢brillary acidic protein in human cerebellum. Acta Histochem. 95, 13^21. Simmons, D.D., Moulding, H.D., Zee, D., 1996. Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study. Dev. Brain Res. 95, 213^226. Sokolowski, B.H., Cunningham, A.M., 1996. Sensory cells of the chick cochlea express synaptophysin. Neurosci. Lett. 216, 89^92. Spoendlin, H., 1979. Sensory neural organization of the cochlea. J. Laryngol. Otol. 93, 853^877.
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Synaptophysin immunohistochemistry in the human cochlea Shaden Ali M. Khalifa a , Ulla Friberg a , Robert-Benjamin Illing b , Helge Rask-Andersen b
a;
a Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital, 751 85 Uppsala, Sweden Neurobiological Research Laboratory, Department of Otorhinolaryngology, Albert-Ludwigs-University Freiburg, 79106 Freiburg, Germany
Received 4 March 2003; accepted 23 July 2003
Abstract Light microscopy and immunohistochemical analyses of a freshly prepared human cochlea, removed at meningioma skull base surgery, were performed with particular emphasis on synaptophysin (SY) reactivity. Synaptophysin, a 38-kDa glycoprotein, is one of the most abundant integral membrane proteins of small presynaptic vesicles and is a useful marker for sites of synaptic transmission of the efferent olivocochlear system in the cochlea. Following fixation and decalcification, cryosections of 30 Wm were prepared. To introduce immunostaining, free-floating sections were exposed to monoclonal SY antibody. Positive SY immunostaining was solely restricted to the neural and sensory structures and did not include supporting cells of the organ of Corti. Dense reaction products were noted around the hair cells, especially at the basal portion of the inner and outer hair cells and their neural poles, as well as around the inner spiral bundle, tunnel spiral bundle, outer spiral bundle and upper tunnel crossing fibers. The majority of spiral ganglion cells stained positively. An intermingling network of thin unmyelinated nerve fibers stained densely, especially at the basal portions of the cochlea. The spiral limbus, inner and outer sulcus cells, basilar membrane, myelinated nerve fibers, spiral ligament and the stria vascularis were unstained. Human cochlea obtained during surgery offers excellent conditions for immunohistochemical analysis. In the basal cochlea in the organ of Corti, outer hair cell area, there may be alterations due to noise trauma from the drilling procedure. 5 2003 Elsevier B.V. All rights reserved. Key words: Cochlea; Organ of Corti ; Synaptophysin; Human; Noise trauma; Outer hair cell; Inner hair cell
1. Introduction Of the two types of hair cells found in the organ of Corti, inner hair cells (IHCs) and outer hair cells (OHCs), IHCs are the primary transducers of sensory information. The role of the OHCs is still under debate, but they seem to protect the IHCs from loud noise and may also modulate the sound signal. IHCs form chemical synapses with dendrites of type I spiral ganglion neurons (SGNs) that constitute 90^95% of the SGNs.
* Corresponding author. Tel.: +46 (18) 6115303; Fax: +46 (18) 6115360. E-mail address: [email protected] (H. Rask-Andersen).
OHCs synapse on the remaining type II SGNs (Spoendlin, 1979). Synaptophysin (SY), a 38-kDa glycoprotein, is one of the most abundant integral membrane proteins of small presynaptic vesicles and is also a major calcium-binding protein (Rehm et al., 1986). The protein is widely distributed at synapses throughout the nervous system, where it is believed to be involved in the exocytosis of stored neurotransmitter. SY-38, a monoclonal antibody directed against the C-terminus of SY, has proved to be a useful marker for sites of synaptic transmission of the e¡erent olivocochlear system innervating IHCs and OHCs, and has been used to map such transmission in various animals and in humans. Numerous animal studies (Sokolowski and Cunningham, 1996; Gil-Loyzaga and Pujol, 1988; Knipper et al., 1995; Simmons et al., 1996; Liberman et al., 1990; Heinrich et al., 1997; Counter et al., 1997), and a few
0378-5955 / 03 / $ ^ see front matter 5 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-5955(03)00228-4
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human studies (Anniko et al., 1989, 1995; Nadol et al., 1993; Nadol and Burgess, 1994; Rask-Andersen et al., 2000), have shown the presence of SY in e¡erent neural structures in the organ of Corti and spiral ganglion. SY immunoreactivity has been proved to be very sensitive to prolonged ¢xation, and of poor quality following formaldehyde and paraformaldehyde ¢xation. Knowledge of antigen decay due to postmortem artifacts is essential for the correct evaluation of immunoperoxidase studies of reduced immunoreactivity (i.e. a decreased number of SY-positive synapses). The present investigation is the ¢rst to analyze the human organ of Corti immunohistochemically in freshly obtained surgical tissue at varying levels from base to apex using serial cryosectioning of the cochlea. The aim of the study was to elucidate if the use of fresh human inner ear tissue for immuno-studies would give better results than postmortem ¢xed tissue. Another aim was to elucidate if surgically induced cochlear hyperstimulation (drilling) prior to ¢xation would a¡ect immunostaining properties by comparing the staining patterns at di¡erent regions of the cochlea. It is known that high noise levels generated during drilling of long duration may cause temporary threshold shifts (TTS) of up to 5^40 dB (Kylen and Arlinger, 1976; Kylen et al., 1977). Such changes may be partly associated with noise-induced morphological alterations which might a¡ect both a¡erent and e¡erent function and thereby immunostaining (Robertson, 1983).
2. Materials and methods 2.1. Patient data This study is based on the analysis of a human cochlea from a 41-year-old woman with a left-sided 3-cm cerebellopontine angle meningioma. Her preoperative hearing level assessed with four-frequency pure-tone average at 500, 1000, 2000, and 3000 Hz levels was normal (12.5 dB). The removal of cochlear tissue during translabyrinthine surgery for cerebellopontine angle meningioma for the purpose of morphological investigations was approved by the human subjects committee at the Uppsala University Hospital, Sweden. 2.2. Immunohistochemistry The patient su¡ered from a cerebellopontine meningioma which was surgically removed by a transcochlear/petrosal route. The inner ear was surgically removed by drilling and the complete cochlea was carefully drilled out and dissected in one piece. The duration of dissection and drilling to secure the cochlea was approximately 10 min. The bony cochlea was immediately transferred to ice-cold ¢xative containing 4% paraformaldehyde, 0.1% glutaraldehyde, and 15% saturated picric acid in 0.1 M phosphate bu¡er at pH 7.4, where it remained for 72 h. Following ¢xation, decalci¢cation was done in two steps. The cochlea was incu-
Fig. 1. Low-power photomicrograph showing SY-positive spiral nerve structures in the osseous spiral lamina (OSL) in a fresh surgically obtained human cochlea (radial section, middle cochlear turn). Transversely cut immunopositive nerve bundles (arrowheads) are seen together with dense staining in the region of the organ of Corti (OC). SL: spiral limbus. Original magni¢cation U140.
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bated in 8% EDTA in phosphate bu¡er (0.1 M, pH 7.4) at 4‡C for 1 week, followed by another 2 weeks in 25% EDTA at 37‡C. This was found to have decalci¢ed the cochlear bone su⁄ciently to make 30-Wm frozen sections. To introduce immunostaining, free-£oating sections were exposed to three pre-incubation solutions : 45 min in 0.05% H2 O2 , 30 min in 1% milk powder, and ¢nally 30 min in blocking bu¡er containing 10% non-immune bovine serum and 0.05% Triton X-100 in phosphate bu¡er, all at room temperature. Subsequently, sections were exposed to monoclonal SY-38 antibody (Boehringer Mannheim, Germany) at a concentration of 0.14 Wg/ml. After an incubation time of 72 h at 4‡C, binding sites of the receptor antibodies were detected using the avidin^biotin technique (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine (0.05%) and ammonium nickel sulfate (0.3%). Finally, sections were mounted on gelatinized slides, dehydrated in a graded series of ethanol, and coverslipped in Entellan (Merck, Germany).
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2.3. Light microscopy (LM) The organ of Corti was sectioned separately from the lower basal, upper basal, lower middle, upper middle and apical regions. The di¡erent turns were analyzed using LM.
3. Results 3.1. Spiral nerve ¢bers Spiral ¢bers appeared in small bundles that were four to six in number. The myelinated ¢bers in the proximal osseous spiral lamina were unstained, while the ¢bers appearing at the transitional zone and peripheral to the habenula perforata showed strong SY immunoreaction (Fig. 1). 3.2. Organ of Corti The radial a¡erents and lateral e¡erents innervating
Fig. 2. Higher-power photomicrograph of the human organ of Corti demonstrated in Fig. 1. SY-positive nerve ¢bers can be seen at the level of the habenula perforata (arrowhead). Positive staining is also seen at the neural poles of IHCs and OHCs. Inner (IP) and outer pillar cells (OP) are unstained. SL: spiral limbus. ISC: inner sulcus cells. Original magni¢cation U490.
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Fig. 3. Adjacent section showing SY staining below and adjacent to the IHCs and OHCs (arrowheads). Some staining probably also occurs in the IHCs. The tunnel spiral bundle (TSB; mediating e¡erents to the OHC region) is positively stained. IPC: inner pillar cell. OPC: outer pillar cell. BM: basilar membrane. V: spiral vein. Original magni¢cation U1200.
the IHCs coursed together and stained strongly SY-positive (Figs. 1 and 2). It was not possible to distinguish between a¡erent and e¡erent ¢bers innervating the IHCs (Fig. 3). Innervation of the OHCs showed strong SY immunostaining of the medial e¡erents, represented by the upper tunnel crossing ¢bers and tunnel spiral bundle (Fig. 4). Upper tunnel crossing ¢bers and tunnel spiral bundle SY immunoreactivity sometimes had a ‘beaded’ or varicose appearance. SY immunoreactivity was equally strong in nerve endings below the OHCs and IHCs (Figs. 1^4). 3.3. Hair cells The hair cells also showed positive SY staining. The basal two thirds of the £ask-shaped bodies of the IHCs were stained with a gradual weakening towards the apex. The nucleus, the cytoplasm below the cuticular plate, and the stereocilia appeared unstained (Fig. 4). The rod-shaped OHCs stained more uniformly throughout the body of the cell, although the cuticular
plate and stereocilia remained unstained (Figs. 3 and 4). 3.4. Supporting cells Inner and outer pillar cells were unstained, as were Deiters’ cells (Fig. 4). 3.5. Spiral ganglion The major part of large spiral ganglion cells (type I) proved SY-positive, while a few showed only faint staining (Fig. 5). One or two appeared unstained. Small ganglion cells (type II) appeared to be SY-positive, although some heterogeneity in staining properties was noted. Myelinated nerve ¢bers were immunonegative. Unmyelinated ¢bers were strongly SY-positive and had a beaded or varicose appearance. These ¢bers intermingled with spiral ganglion cells and a close physical relationship was sometimes noted. The unmyelinated ¢bers merged into a larger bundle constituting the
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Fig. 4. LM of the human organ of Corti at the upper basal region of a fresh surgically obtained human cochlea. The tunnel spiral bundle (TSB) and upper tunnel crossing ¢bers (e¡) are SY-positive. Dense staining products occur below the OHCs and IHCs. Stereocilia and cuticular plate of the IHC can be seen (arrowhead). Some staining also occurs in the cytoplasm of the hair cells. OP: outer pillar cell. IP: inner pillar cell. BM: basilar membrane. V: spiral vessel. Original magni¢cation U700.
e¡erent intraganglionic spiral bundle, which was also strongly SY-positive (Fig. 5). 3.6. Radial topographic variations Radially, the strength of SY staining decreased somewhat from the ¢rst to the third row of the OHCs (Fig. 3). 3.7. Longitudinal topographic variations The SY staining pattern along the basal, middle and apical turns was not uniform in the various cell structures of the organ of Corti. The IHCs and nerve ¢ber structures around the IHCs stained rather equally from base to apex. The OHCs stained more weakly towards the apical region, as did the e¡erent nerve terminals and e¡erent nerve ¢ber structures. Towards the basal region, the OHCs were sometimes clustered into one entity, rendering it impossible to discern the individual OHCs. Towards the apex, the OHCs were seen to gain length and were separated into three or four distinguishable rows (Figs. 3 and 4).
4. Discussion The monoclonal antibody for SY has a⁄nity for identi¢ed presynaptic vesicle glycoproteins and has been used as an intracochlear marker for e¡erents of the olivocochlear bundle. Experimental animal studies have shown a reaction with anti-SY in the sensory cells of the avian cochlea (Sokolowski and Cunningham, 1996). In the developing rat and guinea pig, SY immunolabeling marked the varicosities of the olivocochlear e¡erent endings around IHCs and OHCs (Gil-Loyzaga and Pujol, 1988), and also monitored growth and synaptogenesis of e¡erents in postnatal development of the inner ear in the rat and hamster (Knipper et al., 1995; Simmons et al., 1996). In the cat, SY visualized e¡erent nerve endings at the IHCs and OHCs (Liberman et al., 1990). Strong SY immunoreactivity was present in the e¡erent nerve endings near the base of IHCs and OHCs as well as in the cytoplasm of OHCs in guinea pig studies (Heinrich et al., 1997). Another guinea pig study using quantitative analysis of SY immuno£uorescence demonstrated e¡erent nerve endings at the IHCs to be smaller, more numerous, and more densely packed
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Fig. 5. LM showing SY immunohistochemistry of the human spiral ganglion of the middle cochlear turn of a fresh surgically obtained human cochlea. Most ganglion cells show strong immunopositivity while a few show only faint staining (arrowheads). Myelinated nerve ¢bers are immunonegative while many unmyelinated ¢bers show positive reaction. The unmyelinated ¢bers merged into a larger bundle constituting the efferent intraganglionic spiral bundle (IGSB). These ¢bers are frequently physically related to the spiral ganglion cells. Original magni¢cation U390.
compared to OHC e¡erent nerve endings. This study also showed a longitudinal gradient with a higher percentage of e¡erent terminals innervating the IHCs and OHCs in the basal and mid segments than in the apical regions (Counter et al., 1997). Liberman et al. (1990) noted e¡erents to the OHCs to be most numerous at the basal cochlear turns between the 6- and 24-kHz region, whereas they found e¡erents to the IHCs to be uniformly distributed throughout the cochlea. Earlier studies on the human inner ear showed staining by SY in the apical region of both OHCs and IHCs as well as in adjacent nerve terminals in the fetal inner ear. Adult hair cells were shown to have immunoreactivity throughout the cytoplasm (Anniko et al., 1989). In more recent studies using neonatal and infant human temporal bones, SY was found at the base of the IHCs and OHCs, and in the inner spiral bundle, outer spiral bundle, tunnel spiral bundle and upper tunnel crossing ¢bers (Nadol et al., 1993). Anti-SY reactivity also showed decreasing immunoreactivity from base to apex and from the ¢rst to third OHC rows (Nadol et al., 1993). Using transmission electron microscopy, the same group (Nadol and Burgess, 1994) also showed
labeled e¡erent synapses on Deiters’ cells. Another study on the adult human inner ear showed SY expressed only in spiral ganglion cells, in contrast to the calcium-binding protein S-100 which occurred more generally in the labyrinth (Anniko et al., 1995). Spiral ganglion cells were mostly SY-positive, as were unmyelinated beaded nerve ¢bers and bundles associated with the ganglion cells in preparations of freshly obtained inner ear tissue (Rask-Andersen et al., 2000). The degree of antigen decay due to postmortem artifacts and ¢xation could account for the heterogeneous staining patterns of hair cells in di¡erent studies. Aspeci¢c background staining is known to increase postmortem and SY immunoreactivity and has been proved to be very sensitive to prolonged ¢xation (Sillevis Smitt et al., 1993). In the present study, the use of a fresh surgically obtained cochlea with a short ¢xation and decalci¢cation time minimized SY antigen decay. There was excellent preservation of antigenic sites throughout the cochlea. The SY-positive labeling of neuronal and sensory structures of the cochlea was in accordance with earlier studies on humans. However, at the light microscopic level it was not possible to make distinctions
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between stained and unstained nerve endings below hair cells, which would represent the e¡erent and a¡erent systems, respectively. Fine ¢bers known to represent the olivocochlear system, such as the inner spiral bundle, upper tunnel crossing ¢bers, and outer spiral ¢bers, were strongly SY-positive, indicating that they were efferent ¢bers which couple to the OHCs and synaptic a¡erent terminals of the IHCs. The ¢ndings in the present study regarding di¡erences in staining of IHCs and OHCs could represent nerve endings terminating at the supranuclear region of the OHCs as described by Liberman et al. (1990) and Nadol et al. (1993). The morphology and course of SY-positive nerve ¢bers indicate possible synaptic contacts with spiral ganglion cells. In the present study the IHC staining properties did not show signi¢cant variation longitudinally. This was not in accord with earlier studies by Counter et al. (1997), who showed more e¡erent terminals innervating the IHCs at the basal and middle segments of the cochlea using quantitative analysis of SY immuno£uorescence. The present results were more in accord with those of Liberman et al. (1990), who found e¡erents to the IHCs to be uniformly distributed throughout the cochlea. The OHCs stained more weakly towards the apical region and this was also in accord with ¢ndings of Liberman et al. (1990), who noted e¡erents to the OHCs to be most numerous at the basal cochlear turns between the 6- and 24-kHz region. Earlier transmission electron microscopy studies have revealed that synaptic contact sites are frequently encountered on the small cells (type II) and sometimes even on the large cells (type I) in the human spiral ganglion. Such synaptic contacts have been observed at sites where unmyelinated ¢bers from the e¡erent intraganglionic spiral bundle are in close association with the type II spiral ganglion cells. The positive staining of most of the small ganglion cells is in accordance with previous studies indicating close connections between e¡erents of the olivocochlear bundle and a¡erent neurons within the spiral ganglion suggesting that the OHC system may be under the in£uence of e¡erent innervation at the level of the spiral ganglion (Rask-Andersen et al., 2000). The noise levels reaching the cochlea during drilling of the mastoid process, as measured in human cadavers and temporal bones, showed that the ipsilateral cochlea was exposed to noise levels of about 100 dB (Kylen and Arlinger, 1976). Drilling on the ossicular chain generated vibratory stimulation equivalent to sound pressure levels on the eardrum higher than 130 dB (Helms, 1976). TTSs have been described after ear surgery for chronic otitis media and varied between 5 and 40 dB, with a positive correlation between magnitude of TTS and duration of noise exposure. The TTS was detected
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by means of pre- and post-exposure electrocochleograms. The frequencies primarily a¡ected were 4 and 8 kHz (Kylen et al., 1977). Bone drilling in the meningioma patient in this study was conducted for approximately 5 h, followed by 10 min of drilling directly on the cochlear otic capsule before ¢xation. Such energy levels could theoretically lead to structural noise damage projecting especially around the vulnerable 4-kHz area in the basal turn of the cochlea. To date, no study using anti-SY labeling of the organ of Corti has been performed immediately after noise trauma in experimental animals. One SY study in the guinea pig was performed after a 1-month recovery period. The weak SY activity at the lesion sites was speculated to be due to a reduction in number or size of terminal e¡erent endings or synaptic vesicles. Exhaustion of synaptic vesicle stores after exposure to continuous intense noise stimulation might alter e¡erent ending reactivity (Canlon et al., 1999). Morphological studies in the organ of Corti of experimental animals in general using light and electron microscopy have shown that OHCs are more vulnerable to acoustic trauma than IHCs, and particularly in the basal turn. Nerve endings are more resistant (Lim, 1976). In rats where one ear was exposed to 130 dB for 30 min, signi¢cantly more damage to the transductional apparatus was found at high-frequency regions as compared to low-frequency regions (Michler and Illing, 2002). In rabbits exposed to 100 dB for 2 h, examination in the acute stage, using electron microscopy, showed that the infranuclear region of OHCs had the most damage, and this was related to the degree of change seen in the e¡erent nerve endings (Omata et al., 1992). The e¡erent terminals showed dilated mitochondria and a decrease in number of small vesicles immediately after acoustic exposure (Omata and Schatzle, 1984). The acute changes, however, are mostly reported to be subtle. Chan et al. (1998) reported a reduction of sti¡ness and cell length of the OHCs in the acute post-exposure phase. Nordmann et al. (2000) observed a buckling of the pillar bodies in chinchillas after 24 h of exposure to 86 dB and noted that OHC stereocilia were not embedded in the tectorial membrane at the site of the noise-induced TTS. In the present study the morphology of the organ of Corti in basal turn appeared altered. It is not known whether the cause of these changes, occurring mainly in the OHC region, was a preparational artifact or if the changes re£ect functional modi¢cations due to acoustic hyperstimulation occurring prior to ¢xation. In that case the variances in staining properties may correspond to various frequency regions. The SY labeling in the basal turn, giving the impression of OHCs lumped together, could be in accordance with alterations noted earlier after acoustic trauma in animals,
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e.g. shortening of the OHCs as described by Chan et al. (1998) and buckling of pillar bodies (Nordmann et al., 2000). To draw conclusions from morphological changes in the experimental animal to the human organ of Corti can, however, be dubious and interindividual variations in susceptibility to noise trauma between humans make it hard to predict morphological changes. The study con¢rmed that fresh surgically obtained human cochlear tissue is well suited for immunohistochemical studies. One disadvantage could be that the specimens might be a¡ected by acoustic overload causing cellular and subcellular changes. It is unclear if acoustic stimulation of 100 dB for 5 h is su⁄cient to modify the synaptic and OHC architecture in the human inner ear and whether this may lead to the occurrence of structural damage before the ¢xation. The excellent preservation of antigenic sites could o¡er an opportunity to study how functional modi¢cations may a¡ect results of inner ear immunolabeling.
Acknowledgements The authors wish to thank Anders Kinnefors for the skillful surgical procedure involved in removing the cochlea, and Ingvor Forsberg for skillful technical assistance. The study was supported by grants from the Swedish Research Council Project 3908 and from Stiftelsen Tysta Skolan.
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