Intracellular calcium and outer hair cell electromotility.

The influence of increased intracellular calcium level on outer hair cell (OHC) electromotility was examined by means of transcellular electrical stimulation in a partitioning microchamber. Electromotile activity was measured before and after application of the calcium ionophore ionomycin, which promotes the inflow of extracellular calcium, as well as its release from intracellular calcium stores. The ionomycin solvent, dimethyl sulphoxide (DMSO), by itself elicited a significant decrease in the magnitude of OHC electromotility. The DMSO effect was counteracted by 10 microM ionomycin and was reversed by 50 microM ionomycin. The increase in electromotility is partially mediated by a calmodulin-dependent mechanism, since W7, a calmodulin antagonist, attenuated the 50 microM ionomycin-induced motility increase. Our results suggest that the electromotility magnitude increase in isolated OHCs due to ionomycin is a calcium/calmodulin-dependent phenomenon.

Prognostic significance of P53 histochemistry and DNA histogram parameters in head and neck malignancies.

BACKGROUND: The aim of the study was to determine the role of quantitative pathological parameters in prognosis of head and neck malignancies. MATERIALS AND METHODS: 51 head and neck squamous cell carcinoma patients were examined for mutant p53 gene expression (45 out of 51 patients) by immunohistochemistry and for cellular DNA-content (44 out of 51 patients) using digital picture analyzer. Statistical analysis was performed using BMDP package. RESULTS: No correlation with prognosis was found for age, sex, localization, T-classification and therapy. There was significant relationship between N-status and overall survival (p = 0.0008). No correlation was found with overall and disease-free survival for either histologic type or grading. P53: No significant correlation was detected with overall survival. A relationship was found between mutant p53 and metastasis-free time (p = 0.06). Ploidy: There were no significant differences between aneuploid and euploid tumors for either disease-free or overall survival. Synthetic (S)-phase fraction: A correlation was found for both survival rates (p = 0.029) and metastasis-free time (p = 0.05). Polyploid fraction (PF): correlation was shown for both overall survival (p = 0.0128) and metastasis-free time (p = 0.0038). CONCLUSION: There is correlation between p53 overexpression and metastatic potential and there is a significant relationship between SPF and PF value and prognosis (metastasis-free and overall survival) of head and neck cancer.

Cyclic GMP and outer hair cell electromotility.

The aim of this study is to examine the effect of phosphorylation pathways on the electrically evoked fast motile response of isolated outer hair cells (OHCs). Transcellular electrical stimulation was applied in the microchamber to guinea pig OHCs and motility was measured before and after drug application. Forskolin (adenylate cyclase activator), phorbol 12-myristate 13-acetate (PMA, protein kinase C activator) and dibutyryl 3',5'-cyclic guanosine monophosphate (cGMP agonist) were studied. As controls, L15 medium and dimethyl-sulfoxide (DMSO) were used. In each group, 12 cells were measured. Forskolin and PMA were dissolved in 0.1% DMSO to render them membrane permeable. DMSO by itself caused a statistically significant electromotility magnitude decrease. Forskolin and PMA could not reverse the motility decrease due to DMSO, the effects seen in their presence were the same as observed with DMSO alone. Thus, neither 3',5'-cyclic AMP-dependent protein kinase nor calcium/phospholipid-dependent protein kinase appear to have modulatory effects on electromotility. Dibutyryl cGMP (DBcGMP), in concentrations of 200 microM, elicited a significant electromotility magnitude increase. The DBcGMP effect could be inhibited by co-application of 200 microM DBcGMP and 100 microM 8-Rp-pCPT-cGMPS (8-4-chlorophenylthio-guanosine 3',5'-cyclic monophosphothioate, Rp isomer, a cGMP antagonist). Our results suggest that OHC electromotility is modulated by a cGMP-dependent pathway.

Otoacoustic emissions: a new tool for monitoring intracranial pressure changes through stapes displacements.

Changes in hydrostatic intracranial pressure (ICP) are thought to be transmitted to cochlear liquids, thereby altering the mechanical load on the stapes footplate. Hence the stiffness of stapes' annular ligament is expected to reflect ICP changes. Corresponding middle-ear transmission changes have been assessed using click-evoked otoacoustic emissions (EOAE) in two experiments. The first one was performed in 22 normal human subjects submitted to posture changes. In the second one, controlled ICP increases were applied to 18 patients in neurosurgery operating theater. EOAEs were monitored in these subjects throughout the experiments and their phases and amplitudes were analyzed as a function of frequency. ICP affected EOAEs in a systematic manner in both experiments, i.e., the main effect of ICP increase was phase lead of EOAE components below 2 kHz without any significant amplitude or frequency shift. Their variations were much more evident than the modifications in middle-ear impedance assessed by standard impedance or admittance measurements. The second experiment led to a quantitative linear relationship between ICP and EOAE phases. It demonstrated that the sensitivity of EOAE phase monitoring technique proves to be quite high, i.e., 55 daPa increases in ICP are readily detected in all subjects. The results are consistent with the predictions of classical middle-ear models as to the transmission alterations induced by ICP-related changes in the stiffness of the annular ligament of the stapes. Non-invasive monitoring of ICP in patients with hydrocephalus treated with ventriculo-peritoneal shunts might be attempted with this technique, although it is restricted to patients with detectable EOAEs (i.e., about 50% in this series).

Air caloric computer system application in monitoring vestibular function changes after cochlear implantation.

INTRODUCTION: The computer-based ENG system's analytical routine refinements are available only in research laboratories. The computer-based system contains a stimulator for saccadic eye-movement and an air caloric stimulator interconnected to the registration program and analysis software. Several authors have reported the preoperative and postoperative vestibular functions in cochlear implant patients. The safe examination of the operated ear and comparison of the preoperative and postoperative average slow phase velocities of the caloric nystagmus is possible using the air caloric computer system. METHODS: The authors have used a computer-based ENG system with caloric air stimulation, which is very useful for examination of the operated ear. The vestibular system of patients with total deafness was examined before and after the cochlear implantation. The results of 60 vestibular examinations of 64 patients are reported. RESULTS: The vestibular function in the operated ear was found unchanged in 20 patients. In 14 patients the vestibular function was worse. In 16 patients the postoperative vestibular responsiveness improved. The detailed data analysis of 10 patients in whom preoperative and postoperative vestibular examination was similar was performed by the authors. The preoperative and postoperative average slow phase velocity values and the relative canal paresis was observed. The values demonstrated that the caloric responsiveness of the operated ear improved in few cases. CONCLUSIONS: The computer-based air caloric system is a useful and safe method in evaluation of the vestibular system changes after ear surgery. The reason of the improvement of vestibular responsiveness is not clear. The results need further investigation to solve the problem of vestibular responsiveness improvement after cochlear implantation.

Examination of noncollagen protein composition of the footplate and superstructure of the otosclerotic stapes.

We have differentially precipitated 2 fractions of noncollagen proteins extracted from separately pooled superstructure and footplate of surgically removed stapes of patients from both sexes, who have suffered from otosclerosis. The two fractions were: EDTA-soluble, CaCl2 precipitable (Fraction-1) and EDTA-soluble, CaCl2 non-precipitable proteins. The protein pattern of these two fractions was compared by isoelectric focusing and two-dimensional gel electrophoresis. The phosphoprotein fraction (Fraction-1) of the otosclerotic stapes footplate contained acidic (pI 3-6), low molecular weight (20-40 kD) proteins, which were not detected in the superstructure and temporal cortical bone. Two-dimensional mapping of the Fraction-2 showed predominantly acidic proteins in the footplate and some basic minor components in the superstructure. The lack of the low molecular weight proteins in the superstructure proves the localisation of the otosclerotic process only in the footplate.

Cathepsin D activity in otosclerotic bone and perilymph.

Otosclerotic bone and perilymph samples obtained during stapedectomy operations from patients suffering from pure conductive hearing loss of at least 40 dBs air-bone gap at 500 Hz. Control perilymph samples derived from patients suffering from round window membrane rupture or Meniere's disease. Enzyme activities were determined using a synthetic fluorogenic substrate by fluorescence spectrophotometry. No significant differences have been found between the protein content of otosclerotic and normal or non-otosclerotic perilymph samples. Cathepsin D activity was significantly higher (p less than 0.001) in the otosclerotic perilymph samples. Otosclerotic stapes footplate contained more activity than normal meatal cortical bone as well, though the difference was not significant. Biological significance of the elevated Cathepsin D activity must be considered.

Effect of flavonoid on PGE2-induced alterations in percentage collagen synthesis in ossicle organ cultures.

In adult guinea-pig stapes organ cultures, 3H (2,3)-proline incorporation into the collagenase-digestible protein (CDP) and non-collagen protein (NCP) fractions of the ossicles was measured and the percentage of collagen synthesis (PCS) was calculated. Prostaglandin E2 (PGE2), a potent stimulator of bone resorption, inhibited the PCS in low concentrations (5 and 25 microM), whereas it stimulated it in pharmacological concentrations (50 and 100 microM). Ipriflavon, an isoflavone derivative of therapeutical potential in otosclerosis, also reduced the PCS in 1, 10 and 50 microM concentrations. 50 microM Ipriflavon stimulated the PCS inhibited by 5 microM PGE2, but decreased the PGE2-induced PCS enhancement in vitro.

Cochlear implantation improves hearing in the contralateral ear.

The electrical stimulation of deaf ears by cochlear implants (CI) improved contralateral hearing in seven patients: 5 intracochlear (IC) (analogue processor) and 2 extracochlear (EC) (digital processor) implants. Our patients obtained best speech discrimination scores (98% + 2%, n = 11) when lip reading and CI were combined with a high-power hearing aid fitted into the contralateral ear. Contralateral hearing improvement was 10-15 dB sound pressure level (SPL) at frequencies up to 4000 Hz; hearing in a previously totally deaf ear recovered 4-6 months after CI. The phenomenon was independent of the type of speech-encoding (analogue or digital). Five out of 6 reimplanted patients who first received an 8-channel digital-pulsatile EC reported better speech discrimination after the second CI, when IC devices were implanted with analogue speech processors. These results suggest that EC implantation should be considered in childhood as a first choice, since i) It does not exclude the possibility of a second IC implantation with the same success rate as for the first; ii) The inner ear sensory epithelium remains intact after EC implantation, and consequently, if any regenerating process takes place in the implanted ear due to electrical stimulation it may provide more physiological hearing later on.

Flavonoids alter bone-remodelling in auditory ossicle organ cultures.

The beneficial role of bioflavonoids in an otosclerosis-like bone-remodelling process can be implicated from its interference with bone resorption induced by prostaglandin E2 (PGE2) in cultured guinea pig ossicles. Ipriflavone (7-isopropoxy-isoflavon) and quercetin reduced PGE2-elevated collagenase-like peptidase (Cl-peptidase) activity and potentiated a PGE2-induced decrease in collagen synthesis. The fact that PGE2 effects are mediated through cyclic AMP in bone turnover and flavonoids act synergistically with PGE2 in collagen synthesis confirm a cyclic AMP phosphodiesterase inhibitory role of flavonoids. It has already been attempted to use Ipriflavone medical treatment of otosclerosis. Quercetin, which has a better than Ipriflavone water-solubility seems as promising as Ipriflavone in the control of the otosclerotic bone-remodelling disturbance.

Oversulfated mucopolysaccharides in the otosclerotic bone.

Mucopolysaccharides (glucosaminoglycans, GAGs) were extracted from otosclerotic and non-otosclerotic human stapes footplate, stapes superstructure and guinea pig stapes footplate. Cellulose acetate electrophoresis revealed an unknown highly anionic band (Rf = 1.05) beside the conventional hyaluronic acid, chondroitin sulfate and heparan sulfate fractions. Chemical analysis resulted in a high sulfate/hexosamine molar ratio in the otosclerotic GAG extract (1.7 and 1.9) and a very low ratio in non-otosclerotic human stapes and normal guinea pig stapes footplate GAG extract (0.20 and 0.21, respectively). Hexuronic acid content was highest in guinea pig stapes GAG preparation. Our results serve as indirect evidence of the presence of an oversulfated GAG fraction in the otosclerotic bone, which can potentially initiate otosclerotic bone resorption.

Clinical and experimental studies on the incorporation of materials applied as covering for the labyrinth window.

From among the materials used for covering the labyrinth window, of the foreign materials gelfoam proved ideal from clinical and experimental points of view, while of the materials of interstitial origin, fascia, perichondrium, and periosteum seem to be suitable for covering the labyrinth window, resp. for transplantations in the tympanic cavity.

New method for inserting and fixing the active electrode during cochlear implantation.

Cochlear implantation has been performed for 15 years by the authors; altogether 117 implantations were carried out during this period. Different techniques had been used: at first after having performed atticoantrotomy the active electrode was inserted through posterior tympanotomy but fixing the electrode with this method was relatively not that easy. During the last 6 years implantations were carried out using the authors' new method. At the beginning of the operation cochleostomy was prepared by means of lateral tympanotomy. Introduction of the electrode was performed through a tunnel that had been drilled previously through the posterior wall of the external auditory canal. With a piece of muscle the active electrode was properly secured in the cochleostomy and also a narrow tunnel was prepared in the posterior wall for securing the cable with a Dacron tie. The authors' method of implantation proved to be perfect for introducing the electrode and also for fixing it with long-lasting results.

Reoperations after cochlear implantation.

During the 14-year history of cochlear implantation at Semmelweis University, 95 implantations were performed. There were 15 reoperations, and 2 patients had been operated 3 times. All of the reoperated patients received round window or intracochlear MedE1 devices. Successful reinsertion of the electrode was performed without significant difficulty, though in some cases osteoneogenesis was found in the round window or in the scala tympani. None of the patients suffered any inner ear abnormalities due to the first implant, and even the patient with a third implant in the same ear had excellent results and obtained better discrimination scores with the new device. In the authors' opinion, on the basis of the accumulated experience of nearly 100 implantations and 15 reoperations, replacement of the early generation extracochlear implants is most advisable and can be accomplished without adverse effects.

Hearing improvement achieved by different cochlear implantation techniques on the same and contralateral ears.

The authors have performed cochlear implantation since 1985. Different implantation techniques have been used: extracochlear, round window and intracochlear implantation combined with different receivers. Right at the beginning, the 8-channel extra-cochlear implant produces good results due to the gradually increasing current needed to obtain the same effectivity (transduction energy). However, no great differences can be observed in speech reception whether round window or intracochlear implants be used. In addition to audiological results, we briefly outline surgical implantation techniques and implant fixation methods and summarize the outlook for long-term cochlear implantation. Six months after operation, contralateral hearing was observed. The hearing presented itself at one or two frequences.

Comparative results with different cochlear implants.

The cochlear implant program in Budapest began in 1985, since when 60 operations have been performed, 14 of them on children (51 primary procedures and 9 reimplantations). Different devices and also different techniques have been used: extracochlear promontory, extracochlear round window and intracochlear implants. Various speech processors were applied; at first a digital pulsatile sound-encoding system, later on analog processors were used, while processors operating on the basis of the CIS strategy are preferred nowadays. The operations were performed on both pre- and postlingual patients. In several cases contralateral hearing improvement was observed 6 months after the operation. Considerable experience has been gained of unique cases, such as a deaf-blind prelingual child and prelingual twins. Evaluating cochlear implant performance, in addition to the usual audiological tests measuring postoperative speech understanding, warble tone sound field thresholds were also established.

Possible role of peptides derived from otosclerotic bone in the mechanism of sensorineural hearing loss.

Otosclerotic stapes footplates, superstructures and temporal cortical bones were extracted with 0.25 M guanidine X HCl 0.5 M EDTA (pH 7.4) solution. The extracted non-collagenous peptides/proteins were separated chromatographically on a Sephadex G-25 microcolumn. The peptide composition of the bone samples were compared by capillary analytical isotachophoresis (ITP) in the molecular mass range 0.3-5 kD. The otosclerotic stapes footplate contained 13 ITP subfractions, while the stapes superstructures and cortical bone contained only 9 and 10, respectively. An otosclerosis-specific ITP subfraction was also detected in the stapes footplate, but not in the stapes superstructure or cortical bone. This subfraction was previously demonstrated in the otosclerotic perilymph as well. Four ITP subfractions occurred common in the otosclerotic stapes footplate, the superstructure and the cortical bone. Two of these common subfractions were not found in the cortical bone peptide extract, but all of them revealed higher than normal levels in the otosclerotic perilymph.

Non-collagen proteins of stapedial bone matrix in perilymph of otosclerotic patients.

Non-collagen proteins extracted from the otosclerotic stapes footplate, superstructure and temporal cortical bone were compared with the protein patterns of normal and otosclerotic perilymph by analytical isotachophoresis (ITP). Normal and otosclerotic perilymph yielded basically identical isotachophoretic subfractions. Thirteen subfractions were detected moving as anions at pH 9.6 in the normal perilymph, versus 16 subfractions of similar character in the otosclerotic perilymph. Of these 16 protein subfractions, one (which was not detected in the normal perilymph) could be found in the ITP-gram of the stapes footplate non-collagen proteins, but not of the stapes superstructure or temporal cortical bone. Our results support the concept that protein can enter the inner ear fluid spaces from the otosclerotic bone. The biological significance of these proteins has not yet been clarified.

Cochlear implantation influences contralateral hearing and vestibular responsiveness.

A cochlear implant program has been carried out at the ORL Clinic of Semmelweis University in Budapest since 1985. Different devices and techniques have been used in pre- and postlingual children and adults. Over the last 6 years contralateral hearing improvement has been observed in 18 patients. This phenomenon can usually be demonstrated 6 months after the operation. Since our first observation several other authors have confirmed this phenomenon. However, the underlying mechanism is still obscure; both the efferent innervation and the plasticity of the brain may be important factors. The vestibular function of cochlear implant candidates was checked routinely pre- and postoperatively and changes in vestibular responsiveness were observed. Over the last 3 years air-caloric stimulation has been performed by means of a computer-based ENG system. In most patients the vestibular function was unchanged after the implantation. but in some cases a significant improvement in vestibular responsiveness was noted. Here we briefly describe cases of re-implantation carried out at the clinic. All the patients obtained better speech discrimination scores after having been implanted with intracochlear devices. None of the patients suffered any inner ear abnormality due to the first implant.

Peptides of the otosclerotic perilymph examined by analytical isotachophoresis.

Perilymphs of normal and otosclerotic origin were separated chromatographically on a Sephadex G-25 microcolumn. Peptide composition of the perilymphs was compared by capillary analytical isotachophoresis in the molecular mass range 0.3-5 kD. Otosclerotic perilymph samples contain a heterogeneous, UV-absorbing peptide subfraction which is not detected in the normal perilymph. Normal and otosclerotic perilymph, furthermore, contain four common subfractions detected in twice the normal concentration in the otosclerotic perilymph. These ITP subfractions are degraded during acid hydrolysis (6 M HCI). On the contrary, otosclerosis is a deficient state compared with the normal, as the number of peptides or oligoglycopeptides is twice as high in normal as in otosclerotic perilymph, beside the otosclerosis specific peptides.