IEB 34th meeting Bari: Drugs / Ototoxicity

Oral 18

Cisplatin induced hearing loss. Influence of mode of administration in the guinea pig.

Ekborn A., Laurell G. and Andersson A.

 Cisplatin is an important anticancer drug used particularly against solid tumors of the ovary and testis. Hearing loss is a dose limiting  side effect, especially in palliative treatment. This study was performed to determine the influence of the mode of drug administration and plasma pharmacokinetics on the ototoxic side-effect. Cisplatin (8 mg/kg body weight) was given intravenously to guinea pigs either as a 15 second bolus injection (20 animals) or as a one hour infusion (23 animals). The concentration of free cisplatin was determined in plasma ultrafiltrate using liquid cromatography in combination with post column derivatisation. Electrophysiological hearing thresholds were determined before and 96 hours after drug administration with a standard computerized equipment. The peak plasma concentration in the bolus injection group and in the infusion group was 26 ± 1 µg/ml and 6.2 ± 0.3 µg/ml (mean ± SD), respectively. The area under the plasma concentration curve was not significantly different between the two groups. A significant ototoxic effect evaluated as shift of electrophysiological hearing threshold was observed in both groups (p<0.001 at 20 kHz and p<0.01 at 12.5 kHz in both groups) but there was no significant difference between the two groups (p>0.2). The animals in the bolus group did sustain a weightloss which was significantly greater (p<0.05) than in the infusion group. A positive correlation between threshold shift and plasma creatinine level was found. It is concluded that the ototoxic effect of cisplatin is not correlated to differences in peak plasma concentration.

Key words:  cisplatin,  plasma pharmacokinetics,  ototoxicity,  nephrotoxicity.

Oral 19

Expression of GAL-1 in the adult rat cochlea after amikacin administration.

Visitación Bartolomé M.¹, Gil-Loyzaga P.¹, Vago P.², Joubert-Caron R.³, Pujol R.² and Lenoir M.²

1Centro de Cultivos Celulares (CAI-UCM) and Dept. de Cirugía (ORL), F. Medicina, U.C.M., Spain.
2INSERM U. 254, Lab. Neurobiol. Audit. Hôpital St. Charles, Montpellier, France.
3Lab. Bioch. and Tech. Prot. U.F.R. Sant., Bobigny, France.

 Hair cells are the primary site of aminoglycoside ototoxic damage.  However, the progressive evolution also involved to the other epithelial cells (Raphaël and Altschüler, 1991).  Some cochlear epithelial cells express, from development until the adulthood, a brain lectin GAL-1, specific for ß-galactosides structures, of the cochlea (Remezal et al., 1993).  The aim of the present study was to analyze the expression of GAL-1 in cochleas of adult rats which received amikacin treatment in the juvenile period.
 In this study 21 Wistar rats received a daily intraperitoneal injection of 500mg/kg of amikacin (Bristol) between PND 9 and 16 (Lenoir and Vago, 1996).  Then, they were sacrified, under deep general anesthesia (chloral-hydrate 300mg/kg b.w.), at different ages 21, 28, 45, 70 days.  Both cochleas, rapidly removed were fixed by perilymphatic perfusion in 2% acetic acid in 70% ethanol, and they were embedded in parafin.  Sections, incubated in a 1/1500 anti-GAL1-biot antibody solution (Remezal et al., 1993), were revealed using the avidine-biotin peroxidase method (Vectastain, Vector, Laboratoires Inc.).
 Present result demonstrated that anti-GAL-1 is a suitable antibody to analyze epithelial reorganization in the organ of Corti after ototoxic damage.

Acknowledgements:  supported by grants from the Spanish FISss (95-1540), Human Capital and Mobility (ERBCHRXCT940659) and Acción H.F.231-B.

Key words:  amikacin,  lectin,  ototoxicity,  rat.

Oral 20

Nitric oxide induces apoptosis in the hair cells of cochlea.

Yamane H.¹, Takayama M.¹, Shibata S.¹, Nishimura K.¹, Sunami K.¹, Iguchi H.¹, Konishi K.¹, Nakai Y.¹ and Nakagawa T.².

¹Department of Otolaryngology, Osaka City University Medical School, Osaka, Japan.
²Department of Otolaryngology, Yodogawa Christian Hospital, Osaka, Japan.

 It has been thought that Nitric oxide (NO) has a neuronal cell toxicity under some pathological conditions, especially  in the case of ischemia.  NO is a free radical which can be harmful to living organs, sometimes leading to cell death.  In this study we investigated the effect of NO on the inner ear hair cells of the guinea pig cochlea.  NOC 7, NO donor was placed on the round window membrane of guinea pigs and the cochleas were observed for 78 hours. As a result the outer hair cell death was recognised and its number increased over time .  Cell death was due to apoptosis as shown by examination with transmission electron microscope.

Key words:  inner ear,  nitric oxide,  cell death,  apoptosis.

Oral 21

Localization of megalin, a putative aminoglycoside transporter, in guinea pig cochlea.

Zajic G. and Schacht J.

Kresge Hearing Research Institute, University of Michigan, Ann Arbor MI 48109-0506 USA

 An unresolved question of aminoglycoside toxicity is the organ-specific action against inner ear and kidney and, within the inner ear, the preferential destruction of hair cells. A potential reason for this phenomenon may be a selective aminoglycoside uptake or endocytotic mechanism. Megalin is a glycoprotein expressed in epithelial cells and is engaged in receptor-mediated endocytosis. It is a receptor for polybasic drugs including aminoglycosides, and its localization to renal proximal tubules prompted speculation (Moestrup et al. 1995) that this represents a primary route for aminoglycoside uptake in vivo. However, megalin is also found in tissues that are not susceptible to these drugs.
 Megalin is present in the inner ear of the embryonic mouse (Kounnas et al. 1994) but Ylikoski and collaborators (IEB 1996; ARO 1997) reported that it is absent from sensory cells of the rat cochlea.
 We investigated megalin in the adult guinea pig cochlea by immunocytochemistry. Using peroxidase staining for visualization, antibody was localized to lateral wall tissues, spiral ganglion cells, epithelial cells of the inner and outer sulcus and both supporting and sensory cells of the organ of Corti. The distribution of megalin in hair cells was further investigated by fluorescence laser confocal imaging and by transmission electron microscopy using immunogold labeling. Both procedures showed immunoreactivity associated with stereocilia and the cuticular plate of inner and outer hair cells.
 In summary, megalin remains a candidate for a cochlear aminoglycoside transporter. Its distribution, however, does not match the pattern of ototoxicity. Therefore, different mechanisms must be responsible for the tissue- and cell-selective action of these drugs.

Acknowledgement:  supported by NIH grant DC 000124.

Key words:  aminoglycosides,   uptake,  drug transport.

Oral 22

Formation and calcium incorporation of giant otoconia of guinea pig after streptomycin intoxication.

Harada H., Zhang D.M. and Takumida M.

Department of Otolaryngology, School of Medicine, Hiroshima University, Hiroshima.

 The mechanisms for the formation and fate of giant otoconia following streptomycin (SM) intoxication have been investigated in adult pigmented guinea pigs using scanning electron microscopy.  Calcium turnover into otoconia has also been studied by the use of tetracycline as a tracer.  The administration of SM induced the reduction of otoconia with the formation of giant otoconia.
 The giant otoconia were formed as multifaceted morphology in their early developmental period.  This type of otoconia manifested entire fluorescence indicating existence of calcium uptake.  They then grew up to the transitional type and finally to the cylindrical type.  The giant otoconia is suggested to be formed mainly by dissolution of normal otoconia due to the loss of environmental calcium followed by recrystallization as giant crystals.  The transitional type of giant otoconia had less calcium ion uptake and the removal of calcium from the giant otoconia caused quick disappearance of giant otoconia.  These phenomena might be closely related to the otoconial dynamics which may regulate calcium ion homeostas of endolymph.

Key words:  otoconia,  pathology,  calcium incorporation,  streptomycin,  inner ear,  guinea pig,  tetracycline,  SEM.

Oral 23

Three new synthetic analogues of kynurenic acid affect auditory function.

Gil-Loyzaga P.¹, Hernández E. ², Carricondo F. ¹ and Molina M.T.².

¹Centro de Cultivos Celulares (CAI-UCM) and Dept. de Cirugía, ORL, UCM, Madrid, Spain.
²Instituto de Química Médica (C.S.I.C.), Madrid, Spain.

 Glutamate (GLU) is the most likely neurotransmitter for synapses between inner hair cells (IHCs) and type I afferent dendrites primary auditory neurones (Eybalin, 1993; Pujol et al., 1993; Puel, 1995).  The depolarising effects of GLU goes through at least three types of ionotropic glutamate receptors: NMDA, AMPA and kainate (KA) (Puel et al., 1996).  But GLU, besides its role as excitatory neurotransmitter also has excitotoxic effects (Pujol, 1993).  In this way, the administration of GLU (Jensen, 1988; Janssen et al., 1991; Schweitzer et al., 1991; Janssen, 1992; Gil-Loyzaga et al., 1993; Simón et al., 1994) or its agonists (Pujol et al., 1985, 1995, 1996; Puel, 1991, 1992, 1994; Gil-Loyzaga et al., 1993) induced the swelling of the majority of afferent fibers below IHCs as also happened during hypoxic-ischaemic processes (Puel et al., 1994; Pujol et al., 1992).  Several NMDA and non-NMDA antagonists have been tested to prevent such damage.  The administration of kynurenic acid (KYNA), at low concentrations could be a protective substance (Janssen, 1992).  Even though KYNA also reduced the compound action potential (CAP) (Bobbin and Ceasar, 1987) in particular at high concentrations (Janssen, 1992).  In the present report the effect of three structural analogues of KYNA, QM-96204, QM-9625 and QM-96190 (para- and meta- benzoyl alanine substituted), which were synthesized at the Institute Medicinal Chemistry (C.S.I.C.), were tested.
 Thirty Long-Evans adult male rats were used.  CAP were recorded from a silver electrode, placed onto the round window.  Sound stimuli used were 22, 18, 16 and 12 kHz filtered clicks having 10 ms of duration and the maximum intensity was 90 dB (SPL).  Artificial perilymph, alone or as solvent for drugs (see above), was perfused at a rate of 1.7 µl/min through the scala tympani and allowed to flow out of the cochlea by a second hole made in the scala vestibuli.  After perfusion electrophysiological recordings were performed, and then cochleas were fixed in situ with 2.5% glutaraldehyde in 0.1 M PBS, embedded in Spurr, and morphologically analyzed.  Different results were obtained indicating a neurotoxic action of QM-96204 and QM-9625, while QM-96190 exhibited a main excitatory function.  QM-96204 and QM-9625 induced an increase in the latency and a decrease of the amplitude of the wave of the CAP while QM-96190 reduce the latency and increase the amplitude of the same wave.

Acknowledgement:  supported by the grants from the Spanish FISss 95/1540 and the Human Capital and Mobility ERBCHRXCT940659.

Key words:  glutamate receptors,  kynurenic acid,  kynurenic analogues, cochlea.

Oral 24

Cisplatin ototoxicity and the possibly protective effect of  stimulating hormone () in guinea pigs.

Heijmen P.S., Klis S.F.L., de Groot J.C.M.J. and Smoorenburg G.F.

Hearing Research Laboratories, Department of Otorhinolaryngology, University Hospital Utrecht, Utrecht, The Netherlands.

 Neuro- and ototoxicity are dose-limiting side effects of cisplatin (cis-diaminedichloroplatinum II) cancer therapy. Neurotoxicity can be delayed or prevented by simultaneous treatment with melanocortin-derived peptides such as ORG 2766 (synthetic) and  (natural). Recently, our group has found that cisplatin-induced ototoxicity can be reduced by co-administration of ORG 2766 in guinea pigs (Hamers et al.,(1994), Eur Arch Otorhinolaryngol. 251:23-29; De Groot et al., (1997) Hearing Res. 106:9-19). The present study was designed to investigate the possibly protective effects of  upon cisplatin ototoxicity and to compare the effects of  to those of ORG 2766. Guinea pigs were injected (i.p.) with 2 mg/kg cisplatin for 8 consecutive days;  (75 µg/kg), ORG 2766 (75 µg/kg), or saline (controls) was given (s.c.) immediately before the cisplatin injection and an extra dose was given on day 9. Electrocochleography (compound action potentials, cochlear microphonics) and hair cell counts were performed. Cisplatin treatment and saline co-administration caused severe hearing loss (± 60 dB at 8 kHz) combined with basal and middle turn outer hair cell loss in 5 out of 6 animals. In contrast, in both the ORG 2766 and the  co-treated groups, 3 out of 6 animals could be classified as normal. We conclude that the effects of  and ORG 2766 co-treatment are comparable and that these agents might be clinically useful in protection against cisplatin-induced ototoxicity.

Key words:  cisplatin,  melanocortins,  ototoxicity.

Oral 26

Cochlear hair cells are protected by GDNF against ototoxins.

Magal E., Kuang R., Hever G., Collins F. and Louis J.-C.

Department of Neuroscience, Amgen Inc., Thousand Oaks, CA 91320, USA.

 Sensorineural hearing loss results from the degeneration of hair cells and/or auditory neurons in the cochlea of the inner ear. Aminoglycoside antibiotics, such as neomycin (Lerner et al, N. Engl. J. Med. 302: 1106-1109, 1980), and anti-neoplastic drugs, such as cisplatin (Strauss et al., Laryngoscope, 143: 1263-1265, 1983), are known ototoxins, that cause damage to hair cells and auditory neurons in humans and animals. We found that GDNF protected adult rat auditory neurons and postnatal rat cochlear hair cells in vitro from toxicity induced by neomycin and cisplatin (Magal et al. Neurosci. Abstr. 22: 1621, 1996). We next tested the efficacy of GDNF in animal models of ototoxicity. Adult guinea pigs were pre-treated with GDNF in one ear delivered either directly into the inner ear (infusion into the scala tympani with Alzet mini-pump containing 50 ng/ml GDNF at a rate of 0.5 µl/h), or injected into the middle ear through the tympanic membrane (120 µl at a concentration of 1 mg/ml). In each animal, the other ear served as control. Two days following GDNF treatment, ototoxicity was induced systemically by several paradigms: a) intraperitoneal injections of cisplatin (either at 1 mg/kg/ day for 15 days or two injections of 7.5 mg/kg  at a 5-day interval) and b) combination of kanamycin (200-300 mg/kg administered subcutaneously) and ethacrinic acid (40 mg/kg, intravenous). The experiments were terminated after one month (GDNF inner ear delivery) or after 10 days ( middle ear delivery) and phalloidin-FITC positive hair cells in the organ of Corti were counted in both ears. We found GDNF to be protective against ototoxicity in most of the animals studied regardless of the mode of induction of otototxicity or GDNF delivery. There was a substantial variability between the animals in the degree of toxicity and protection, but in most of them, there were at least 50% more hair cells in the GDNF- treated ear compared with the control ears.

Acknowledgement:  sponsored by Amgen Inc.

Key words:  hair cells,  ototoxins,  GDNF,  protection.

Oral 27

Neurotrophic factor therapy for cochlear nerve degeneration.

Ylikoski J.¹, Pirvola U.² and Saarma M.²

¹Dept. of ORL, University of Helsinki, Finland.
²Institute of Biotechnology, University of Helsinki, Finland.

 Neurotrophic factors (NTFs) are a group of proteins able to regulate development, plasticity and maintenance of structural integrity of neurons.  NTFs have been shown in animal model system to have a neuroprotective role in chronic neurodegenerative diseases and in acute nerve injury which cause structural damage and reactive plasticity in neurons.  At present, there are ongoing clinical trials with NTFs in different neurological diseases in which NTFs are believed to reverse atrophy of neuronal somas and their synaptic contacts.  NTFs are typically synthesized by target cells, bound to specific receptors, internalized in synapses and retrogradely transported to neuronal somas.
 The development of NTFs towards clinical use, e.g. for cochlear nerve degeneration, proceeds along a series of distinct steps: 1. Animal experiments should demonstrate the presence of NTFs in target cells (cochlear hair cells) and their specific receptors in spiral ganglion cells not only during development but also in adult animals.  2. NTFs should, in vitro, promote survival and neuritogenesis of cochlear neurons.  3. NTFs should prevent neuronal degeneration and facilitate regeneration in animal deafness model system.  There are about 20 different NTFs of which 5-6 effectively promote neuritogenesis and survival of cochlear neurons in vitro (criterium #2).  However, only a few, e.g. Neurotrophin-3 (NT-3) and GDNF are present in hair cells of adult rat (criterium #1) and their signalling receptors are found in cochlear neurons (Pirvola et al., PNAS 89, 9915, 1992; Ylikoski et al., Hear Res 65, 69, 1993; Ylikoski et al., ARO Abstr. 1996).  We have tested the effectiveness of exogenously applied recombinant NTFs in prevention of cochlear nerve degeneration in the rat deafness model system (criterium #3).  Our preliminary results indicate that GDNF, possibly also NT-3 and BDNF fulfil this criterium and could thus be a candidate for clinical trials aimed to prevent cochlear nerve degeneration.

Key words:  cochlear nerve,  degeneration,  neurotrophins,  GDNF.

Oral 28

Pharmacological Intervention for NIHL.

Henderson D., Hu B.-H., McFadden S.L. and Kopke R.

Center for Hearing and Deafness, University at Buffalo, Buffalo, New York 14214, USA.

 Reactive oxygen species, which are cytotoxic to living tissues, are thought to be partly responsible for noise-induced hearing loss.  In this study R-phenylisopropyladenosine (R-PIA), a stable non-hydrolyzable adenosine analogue which has been found effective in upregulating antioxidant enzyme activity levels, was topologically applied to the round window membrane.  The contralateral round window had an application of normal saline and served as a control.  The animals were then exposed to a 4 kHz octave band noise at 105 dB SPL for 4 hours.  Evoked potential thresholds and distortion product otoacoustic emissions were tested and hair cell damage was documented.  The mean threshold shifts immediately after the noise exposure were 70 to 90 dB at frequencies between 2 k to 16 kHz with the maximum alteration at 4 to 8 kHz.  One day after noise exposure, recovery from hearing loss in the R-PIA treated ears was 10 to 20 dB greater than in saline treated ears.  Four days after noise exposure, the R-PIA treated ears showed 20 to 30 dB more recovery at frequencies between 2 k to 16 kHz.  More importantly, the permanent threshold shifts tested 20 days after noise exposure showed 10 to 15 dB less in R-PIA treated ears.  The outer hair cell losses in the R-PIA treated ears were also less severe than in control ears.

Key words:  reactive oxygen species,  noise induced hearing loss,  glutathione.

Oral 29

Influence of glucocorticoids and hyperbaric oxygen on cell death in noise exposed cochleas of guinea pigs.

Niedermeyer H.P.¹, Funk A.², Lamm K.¹, Gloddek B.¹, Neumann Ch.¹ and Arnold W¹.

¹Department of ENT, Klinkum r.d. Isar, Technische Universität München, Munich, Germany.
²Institute of Pathology, Klinkum r.d. Isar, Technische Universität München, Munich, Germany.

 Glucocorticoids and hyperbaric oxygen are employed in inner ear diseases such as sudden hearing loss, tinnitus, Meničre disease and acute noise damage. The mechanism of these therapeutic tools is not completely understood. In guinea pigs, noise induces level dependant alterations morphologically in accordance with both, apoptosis and necrosis. The accompanying electrophysiologic changes in the inner ear seem to be influenced by therapeutic agents such as glucocorticoids and hyperbaric oxygen. The aim of our study was to look for the kind and distribution of cell death in noise-induced inner ear hearing loss in correlation to the noise level and the influence of glucocorticoids and hyperbaric oxygen on cell death rate. Broad-band noise exposed cochleas of guinea pigs (95, 101, 106, 115 dB SPL, n=4 each group) were investigated by in-situ-nick-translation and TdT-endlabeling. Furthermore, expression of cell cycle regulatory proteins (p53, bcl-2, bax, mdm-2. mcl1) was investigated by immunohistochemistry. Apoptotic activity was observed beginning at 95 dB SPL with increasing cell death activity up to 115 dB SPL. At 115 dB SPL extensive apoptotic activity of outer and inner hair cells, cells of the stria vascularis and T-cells of the limbus was detected. The influence of the above mentioned therapeutic agents on cell death and the relationship to cell cycle proteins will be discussed.

Key words:  guinea pig,  cochlea,  noise,  cell death.

Poster 18
 
Acute cell toxicity of nitric oxide on the inner ear hair cells.

Nishimura K.¹, Yamane H.¹, Takayama M.¹, Shibata S.¹, Sunami K.¹, Iguchi H.¹, Konishi K.¹, Nakai Y.¹ and Nakagawa T.².

¹Department of Otolaryngology, Osaka City University Medical School, Osaka, Japan.
²Department of Otolaryngology, Yodogawa Christian Hospital, Osaka, Japan.

 Nitric oxide (NO) functions regulating the organ properly, but  can be induce cell injury under some circumstances. In this study we investigated the acute effect of NO on the cochlear hair cells of guinea pig. The collected cochleas were treated in the medium with several concentrations of NO for 3 hours and these morphological changes were observed in a light and transmission electron microscope. Not only the outer hair cell but also the inner hair cell death was dose dependently recognized. These results indicate that excess of NO leads to inner ear hair cell death, leading to inner ear dysfunction.

Key words:  nitric oxide,  cell death,  inner ear,  in vitro.

Poster 19

Glutamate inducing the inner ear hair cell death.

Sunami K.¹, Yamane H.¹, Takayama M.¹, Shibata S.¹, Nishimura K.¹, Iguchi H.¹, Konishi K.¹, Nakai Y.¹ and Nakagawa T.².

¹Department of Otolaryngology, Osaka City University Medical School, Osaka, Japan.
²Department of Otolaryngology, Yodogawa Christian Hospital, Osaka, Japan.

 The signal of glutamate from pre-synaptic cells leads to nitric oxide(NO) release from NO synthase of post-synaptic cells, being an important factor as a neuromodulator in the nervous system.  However, the excess of glutamate beyond the physiological range can be harmful to the organ, leading the cell death.  So far the excitotoxic effect of glutamate has been reported in the inner ear, but its morphological evidence has not been enough to estimate the relationship between glutamate and inner ear. In this study we treated the guinea pig cochlea by glutamate on the round window membrane and the  morphological changes of these cochlea was observed for 72 hours. As a results the outer hair cell death was recognized over 24 hours after the treatment.  This result shows the morphological evidence that the excitotoxicity of glutamate can occur in the inner ear under some pathological conditions.

Key words:  glutamate,  inner ear,  cell death,  in vivo.

Poster 20

Induction of NADPH-diaphorase in the stria vascularis by glutamate treatment.

Takayama M.¹, Yamane H.¹, Shibata S.¹, Nishimura K.¹, Sunami K.¹, Iguchi H.¹, Konishi K.¹, Nakai Y.¹ and Nakagawa T.².

¹Department of Otolaryngology, Osaka City University Medical School, Osaka, Japan.
²Department of Otolaryngology, Yodogawa Christian Hospital, Osaka, Japan.

 It has been known that nitric oxide(NO) released from NO synthase are very important factor in regulating the living organ.  The relationship between glutamate and NO has been also an attractive topics. The released glutamate in the inner ear is promptly diminished, but in the some cases the excess of glutamate has been reported.  In this study we investigated the effect of glutamate on the stria vascularis, specifically, with remarks of induction of NADPH-diaphorase in the stria vascularis. The guinea pig cochlea treated with glutamate on the round window membrane showed the upregulation of NO synthase in the marginal cells, suggesting that stria vascularis is an effective organ by  glutamate signals.

Key words:  glutamate,  NADPH-diaphorase,  inner ear,  stria vascularis.

Poster 21

Apoptosis has a predominant function for deletion of sensory hair cells due to aminoglycoside toxicity in the guinea pig vestibular organ.

Nakagawa T.¹, Yamane H.², Shibata S.², Takayama M.², Nishimura K.², Sunami K.² and Nakai Y.²

¹Department of O.R.L., Yodogawa Christian Hospital, Osaka, Japan.
²Department of O.R.L., Osaka City Univ. School of Medicine, Osaka, Japan.

 Aminoglycoside antibiotics induce vestibular disorders as the results of damage to the sensory hair cells as the peripheral sensory organ.  Recently, involvement of apoptosis has been suggested in the process of hair cell loss, but precise mechanisms are not still determined.  Then we examined dose-dependent effects of streptomycin to degeneration of vestibular hair cells associated with apoptosis.
 We used eleven albino guinea pig.  Animals were given buffered solution containing streptomycin, concentrated at 1 mM, 10 mM or 100 mM, directly to the inner ear.  Control animals were treated with a solution without streptomycin.  One day after treatment, all animals were sacrificed.  Following fixation with 4% paraformaldehyde, cryostat sections of ampullar cristae were made.  Then the sections were stained by the modified TUNEL method or Hoechst #33342.
 TUNEL stain showed the occurrence of DNA fragmentation in hair cells after 10 mM or 100 mM streptomycin treatment, and the number of stained nuclei after 100 mM treatment was significantly higher than after 10 mM.  Hoechst stain revealed apoptotic features of hair cell nuclei following 100 mM treatment.  These findings suggest that affected hair cells are deleted by apoptosis even after high doses treatment, and the number of apoptotic hair cells increases depending on exposed doses of streptomycin.

Key words:  apoptosis,  aminoglycoside,  hair cell,  vestibule,  ototoxicity.

Poster 22

Induction of macrophage exudation in the inner ear by treatment of OK432 treatment.

Shibata S., Yamane H., Konishi K., Iguchi H., Nakagawa T., Takayama M., Nishimura K., Sunami K. and Nakai Y.

Department of Otolaryngology, Osaka City University Medical School, Osaka, Japan.

 OK432, a heat- and penicillin-treated lyophilized powder of a low virulent Su strain of streptococcus pyogenes, was injected into the perilymphatic space of guinea pigs in order to determine the kinetics of exudation of inflammatory cells into the cochlea over a 7-day period.  OK 432 induced exudation of many neutrophils and Asialo GM1-positive cells into the scala tympani.  The number of these cells peaked  on day 1 after OK 432 treatment, and then gradually decreased.   At 4 days after treatment, very few of either of these types of cells were observed in the scala tympani.  Asialo GM1-positive cells were confirmed to be activated macrophages.  In ears treated with physiological saline or the additive included in OK432 in control studies, no notable changes in the inner ear were recognized.   These findings suggest that the inner ear can undergo induction of macrophage exudation by OK432 treatment as has previously been observed for other organs.

Key words:  macrophage,  biological response modifier,  OK432,  inner ear.

Poster 23

Reduced expression of glycocalyx sialoglycoconjugates in outer hair cells after chronic aminoglycoside administration.

De Groot J.C.M.J. and Hendriksen E.G.J.

Hearing Research Laboratories, Department of Otorhinolaryngology, University Hospital Utrecht, Room G.02.531, Heidelberglaan 100, NL-3584 CX Utrecht, The Netherlands.

 Chronic systemic administration of gentamicin results in diminished hair cell glycocalyx reactivity for colloidal thorium (De Groot and Veldman 1988 Hearing Research 35: 39-46).  Thorium reactivity of the hair cell glycocalyx is mainly due to the presence of sialic acid residues.  In order to further quantify the effect of chronic aminoglycoside administration, we have studied the labelling densities of gold-conjugated Limax flavus agglutinin (LFA; specific for sialic acid) and wheat germ agglutinin (WGA; specific for N-acetylglucosamine and sialic acid) in ultrathin frozen sections of microdissected basal, middle and apical turns from guinea pig cochleas, treated with gentamicin (100 mg/Kg/day) and neomycin (100 mg/Kg/day) for 5 or 15 consecutive days.  The number of gold particles present on the apical and basolateral membranes of the outer hair cells (OHC) were counted and labelling densities (expressed as the number of gold particles per µm²) were calculated.
 The basolateral membranes of OHC from normal cochleas demonstrated higher labelling densities of LFA and WGA than the apical membranes, irrespective of the turn studied.  Overall labelling densities of LFA demonstrated an intracochlear gradient: [basal]>[middle]>[apical].  This gradient was not seen with WGA.  Labelling densities of WGA and LFA on the basolateral membranes of OHC in the basal turns were reduced by  after 5-day treatment with gentamicin and neomycin, respectively; a further decrease was seen after 15 days.  A reduction in the labelling densities of WGA and LFA on the apical membranes of OHC was observed only after 15-day treatment with either gentamicin or neomycin.
 These quantitative data corroborate our earlier finding that chronic aminoglycoside administration interferes with the synthesis of membranes glycoconjugates in OHC.  Moreover, the abundance of sialoglycoconjugates in the basal cochlear turn - and the fact that aminoglycosides have a high affinity for sialoglycoconjugates - may explain the finding that aminoglycoside-induced lesions are initially observed in the basal turns of the cochlea.

Key words: inner ear, glycocalyx, hair cells, aminoglycoside, ototoxicity.

Poster 24

Functional and ultrastructural study of the action of coenzyme A in cisplatin-induced ototoxia.

Fernandez-Cervilla F.¹, Cańizares F.J.², Martinez M.¹, Ciges M.¹ and Campos a.².

¹Department of Otolaryngology, Faculty of Medicine, University of Granada, Granada, Spain.
²Department of Histology and Cell Biology, Faculty of Medicine, University of Granada, Granada, Spain.

 An experimental study was done in guinea pigs exposed to increasing doses of cisplatin to produce lesions in the hair cells of the organ of Corti. Co-enzyme A was given prophylactically (simultaneously with cisplatin) or therapeutically (after hearing loss was confirmed).
 Functional tests of hearing were done in all animals before and during the experiment with electrocochleography (compound action potentials). At the end of the experiment the animals were killed and the cochleas were removed and processed for transmission electron microscopic examination of the morphological lesions.
 The results correlated functional alterations with microscopic abnormalities. They also provided evidence of the influence of Co-enzyme A in the hair cell.

Key words:  ototoxia,  cisplatin,  Co-enzyme A,  ultrastructural.

Poster 25

Dose-dependent effect of chronic cisplatin administration upon cochlear hair cells.

Cardinaal R.M., Stengs C.H.M., de Groot J.C.M.J., Klis S.F.L., Smoorenburg G.F., Huizing E.H. and Veldman J.E.

Hearing Research Laboratories, Department of Otorhinolaryngology, University Hospital Utrecht, Utrecht, The Netherlands.

 Cisplatin administration results in loss of outer hair cells (OHC) and subsequent degeneration of the organ of Corti. Recent experimental studies indicate that the degree of cisplatin ototoxicity might be dose-dependent, but the results do not compare well because of the large variety in dosage schedules used. Therefore, we have systematically investigated the dose dependency of cisplatin's ototoxic effect upon the guinea pig cochlea.Guinea pigs were treated by daily intraperitoneal injections of cisplatin for 8 days in different dosages, 0.7 mg/kg (12 ears), 1.0 mg/kg (18 ears), 1.25 mg/kg (10 ears), and 1.5 mg/kg (21 ears), respectively.  Cochlear function was measured electrophysiologically 1 day after cessation of treatment, followed by processing of the cochleas for histological examination. The compound action potential (CAP) and cochlear microphonics showed no significant treshold shifts at doses of 0.7, 1.0 and 1.25 mg/kg. At a dose of 1.5 mg/kg, the CAP showed a significant increase in threshold. No significant OHC loss was seen after cisplatin administration at doses of 0.7, 1.0 and 1.25 mg/kg. At a dose of 1.5 mg/kg we observed a statistically significant loss of OHC and degeneration of the organ of Corti in the basal and middle turns. The electrophysiological and the histological results are in close agreement. Our results have established that the ototoxic effect of cisplatin upon the OHC in the guinea pig cochlea is dose-dependent.

Key words:  cisplatin,  ototoxicity,  dosage, guinea pigs.

Poster 26

Cisplatin ototoxicity and the interaction with an ACTH4-9 analog in guinea pigs.

Stengs C.H.M., Cardinaal R.M., Klis S.F.L., de Groot J.C.M.J., Veldman J.E., Huizing E.H. and Smoorenburg G.F.

Hearing Research Laboratories, Department of Otorhinolaryngology, University Hospital Utrecht, Utrecht, The Netherlands.

 Ototoxicity is one of the main side-effects of  cisplatin. In guinea pigs Hamers et al. have shown that the ACTH4-9 analog , ORG2766, provides partial protection against cisplatin (2mg/kg/day; 8 days) ototoxicity. We hypothesized that at lower cisplatin doses all animals co-treated with ORG2766 would be protected. For this experiment we choose two different cisplatin doses (1.0- and 1.5 mg/kg /day; 8 days; groups of 12 animals per dose). Six animals of each, were co-treated with ORG2766  (75µ/kg/day; 8+1 days). On day 10 the effects were investigated electrocochleographically followed by histological examination. With 1.0 mg/kg/day cisplatin we found only minor effects with cisplatin alone. The animals co-treated with ORG2766 were slightly, but not significantly better. Also histologically there was no significant difference in the outer hair-cell count. With 1.5 mg/kg/day, the co-treated group was electrophysiologically significant better than the non co-treated group. Histologically, we found significantly less outer hair cell loss in the co-treated group. These results confirm the previous results by Hamers et al. showing an ameliorating effect of ORG2766 on cisplatin ototoxicity.

Key words:  ototoxicity,  cisplatin,  cochlea protection.