Stavros Hatzopoulos

Cochlear Repair by Transplantation of Human Cord Blood CD133+ Cells to Nod-Scid Mice Made Deaf with Kanamycin and Noise

We investigated the fate of human cord blood CD133+ hematopoietic stem cells (HSC) transplanted intravenously (IV) into irradiated nodscid mice previously made deaf by ototoxic treatment with kanamycin and/or intense noise, to verify whether HSC engraft the cochlea and contribute to inner ear restoration, in vivo. We tested the presence of HLA.DQα1 by PCR, used for traceability of engrafted cells, finding evidence that HSC migrated to various host tissues, including the organ of Corti (OC). By histology, antibody and lectin-staining analysis, we confirmed that HSC IV transplantation in mice previously damaged by ototoxic agents correlated with the repair process and stimulation ex novo of morphological recovery in the inner ear, while the cochlea of control oto-injured, nontransplanted mice remained seriously damaged. Dual color FISH analysis also provided evidence of positive engraftment in the inner ear and in various mouse tissues, also revealing small numbers of heterokaryons, probably derived from fusion of donor with endogenous cells, for up to 2 months following transplantation. These observations offer the first evidence that transplanted human HSC migrating to the inner ear of oto-injured mice may provide conditions for the resumption of deafened cochlea, emerging as a potential strategy for inner ear rehabilitation.

Evaluation of Cisplatin Ototoxicity in a Rat Animal Model

ABSTRACT: The ototoxic effects of cisplatin were evaluated by otoacoustic emissions and evoked auditory responses. A transient otoacoustic emissions protocol indicated no significant ototoxic effects in rats treated intravenously with 7.5 mg/kg/week for 2‐weeks. A chronic 6‐week treatment (2.5 mg/kg/week) monitored by 2F1‐F2 distortion product emissions presented significant SNR alterations in a narrow range of frequencies (5.04–5.66 kHz). An acute treatment of 15 mg/kg, using slow 30‐min intraperitoneal infusion, presented the highest DP and ABR alterations. The SNR at the 2F1‐F2 frequencies 6.34, 7.13, and 7.56 kHz was found significantly decreased, and ABR latency measurements from 8‐kHz burst stimuli verified these alterations.

Evaluation of anesthesia effects in a rat animal model using otoacoustic emission protocols

Anesthesia effects on otoacoustic emission (OAE) recordings were evaluated in a group of 72 Sprague-Dawley rats (mean weight 225+/-20 gr). Two anesthesia dosages (high and normal) and two anesthetic protocols (ketamine-xylazine, ketamine-xylazine-atropine) were tested. Transient evoked OAE (TEOAE) and distortion product OAE (DPOAE) responses were recorded in 10 min intervals, for a total period of 60 min. Analyses of the data with repeated measure models indicated the following: (1) The animals receiving a high dose of anesthesia (cumulative dose 66.6 mg of ketamine and 13.2 mg of xylazine/kg of body weight) presented significant alterations of the TEOAE response level and the signal to noise ratio at 3.0 kHz; (2) the animals receiving a normal dose of ketamine-xylazine anesthesia (cumulative dose 50 mg of ketamine and 10 mg of xylazine/kg of body weight) presented TEOAE and DPOAE responses invariant in terms of time; (3) significant differences were observed in the DPOAE responses from animals anesthetized with ketamine-xylazine and ketamine-xylazine-atropine. The data support the hypothesis that the ketamine anesthesia OAE suppressing mechanism is related to middle-ear mechanics.

Otoacoustic emissions latency difference between full-term and preterm neonates

Transiently evoked otoacoustic emissions (TEOAEs) were recorded from full-term and preterm neonates. The responses were decomposed, by means of an adaptive approximation method, into waveforms of defined frequencies, amplitudes, latencies and time spans. Statistically significant differences in the latency values were found between the tested groups. Differences were also found in the time spans of the TEOAEs components. For the preterm neonates the contribution of long-duration components (i.e. long-time span) was higher. Those components were characterized by narrow frequency band and contrary to the short-time span components their latencies did not depend on frequency. The removal of the long-duration components, from the pool of analyzed data, decreased the latency differences between the tested groups. The results indicate that the origin of the longer latency values for preterm neonates (with a post conceptional age up to 33 weeks) in respect to full-term neonates can be attributed to the presence of long-lasting components. The correspondence, which was found between frequencies of long-duration components and the spectral peaks of spontaneous otoacoustic emissions (SOAEs), suggests that those components may be connected with SOAEs.

The relationship between distortion product otoacoustic emissions and extended high-frequency audiometry in tinnitus patients. Part 1: Normally hearing patients with unilateral tinnitus

Summary Background The aim of this study was to evaluate distortion product otoacoustic emissions (DPOAEs) and extended high-frequency (EHF) thresholds in a control group and in patients with normal hearing sensitivity in the conventional frequency range and reporting unilateral tinnitus. Material/Methods Seventy patients were enrolled in the study: 47 patients with tinnitus in the left ear (Group 1) and 23 patients with tinnitus in the right ear (Group 2). The control group included 60 otologically normal subjects with no history of pathological tinnitus. Pure-tone thresholds were measured at all standard frequencies from 0.25 to 8 kHz, and at 10, 12.5, 14, and 16 kHz. The DPOAEs were measured in the frequency range from approximately 0.5 to 9 kHz using the primary tones presented at 65/55 dB SPL. Results The left ears of patients in Group 1 had higher median hearing thresholds than those in the control subjects at all 4 EHFs, and lower mean DPOAE levels than those in the controls for almost all primary frequencies, but significantly lower only in the 2-kHz region. Median hearing thresholds in the right ears of patients in Group 2 were higher than those in the right ears of the control subjects in the EHF range at 12.5, 14, and 16 kHz. The mean DPOAE levels in the right ears were lower in patients from Group 2 than those in the controls for the majority of primary frequencies, but only reached statistical significance in the 8-kHz region. Conclusions Hearing thresholds in tinnitus ears with normal hearing sensitivity in the conventional range were higher in the EHF region than those in non-tinnitus control subjects, implying that cochlear damage in the basal region may result in the perception of tinnitus. In general, DPOAE levels in tinnitus ears were lower than those in ears of non-tinnitus subjects, suggesting that subclinical cochlear impairment in limited areas, which can be revealed by DPOAEs but not by conventional audiometry, may exist in tinnitus ears. For patients with tinnitus, DPOAE measures combined with behavioral EHF hearing thresholds may provide additional clinical information about the status of the peripheral hearing.

Dose-dependent protection on cisplatin-induced ototoxicity – an electrophysiological study on the effect of three antioxidants in the Sprague-Dawley rat animal model

Summary Background Sprague-Dawley rats were used as an acute cisplatin ototoxicity model to compare the chemo-protective efficacy of 2 sulphur-containing antioxidants (D-methionine, N-L-acetylcysteine) and 1 seleno-organic compound (ebselen). Each putative chemo-protective agent was tested at 3 different dosages in order to assess the influence of dose on auditory preservation. Material/Methods A total of 40 Sprague-Dawley albino male rats were used in the study. Animals were divided into 10 groups, 3 groups of different doses for each protective agent and a cisplatin-treated control group. The animals were weight-matched before drug exposure to ensure similar weights in all groups. Auditory function was assessed with auditory brainstem responses and distortion product otoacoustic emissions at time zero and at 96 hours post-treatment. Results At the post-treatment follow-up no significant threshold change at 8 kHz was found in the D-Met- and NAC-treated groups. All ebselen-treated animals presented significant threshold elevations. At 12 and 16 kHz, only the groups treated with 300, 450 mg/kg of D-Met and 475 mg/kg of NAC presented thresholds comparable to the pre-treatment ABR data. The ebselen-treated animals presented significant threshold shifts and showed the highest threshold elevations. The DPOAE data analysis showed that only the animals from the 350 mg/kg D-met group presented lack of statistical differences between the pre and post recordings. Conclusions Considering the outcome from the ABR and DPOAE analyses together, only the 350 mg/kg D-met group presented a complete auditory preservation against the 14 mg/kg cisplatin administered i.v. Data from ebselen pre-treated Sprague-Dawley albino male rats demonstrate that ebselen dosages up to 12 mg/kg given by i.p. administration lack auditory preservation in this species.

A comparison of audiometric and objective methods in hearing screening of school children. A preliminary study

OBJECTIVE In newborn hearing screening, one exclusively applies objective hearing testing methods--based on evoked potentials and/or on otoacoustic emissions. However, when testing school children, one can consider both audiometric and electrophysiological methods. The choice of methods is determined by the aims of the program. If one wants to detect conductive hearing losses, impedance audiometry seems to be the method of choice. METHODS The aim of this study was to compare test performance measures from audiometric and objective methods (OAEs and impedance audiometry), in the hearing screening of school children. Screening protocols were applied on a group of 190 children of about 12 years of age (6th grade of primary school). RESULTS For a single application of a screening procedure, the best performance was observed in the automated four-tone audiometry, followed by the tympanometry and the TEOAE-based procedures. Screening performance was enhanced using a combination of automated and impedance audiometry. A four-tone audiometry test combined with tympanometry gives a sensitivity of 65%, and the PPV of 46%, which are reasonable values, acceptable for practical use. The use of a TEOAE protocol degrades the overall performance of screening. CONCLUSIONS Screening of school children is feasible with a combination of automated audiometry and tympanometry with time requirements equal to 3 min per subject.

RNA expression induced by cisplatin in an organ of Corti-derived immortalized cell line

Cisplatin [cis-diamminedichloroplatinum(II)] (CDDP) is an organic compound that is widely used for the treatment of a large number of tumors. Its clinical use is limited by the presence of some undesired side effects, like as oto- and nephro toxicity, whose mechanisms of action are not understood. One of the possible CDDP toxicity mechanism seems to involve the generation of reactive oxygen species (ROS), that can impair morphology and function of hair cells (HC) in the organ of Corti. To test this hypothesis we evaluated the effect of CDDP treatment on RNA steady-state levels of 15,000 genes by microarray analysis, using, as a experimental model, the OC-k3 cell line, obtained from the organ of Corti of transgenic mice and constitutively expressing the large SV40 T antigen. We have found overexpression of several genes related to arachidonate mobilization including phospholipase A2, group IV and V, phospholipase A2 activating protein and lysophospholipase I and III, as well as lipoxygenation like arachidonate 12-lipoxygenase and arachidonate 5-lipoxygenase activating protein. In addition, we found significant transcription of genes regulating cell respiration, including cyt c oxidase, as well as genes involved in xenobiotic detoxification and lipid peroxidation such as cyt P450, and other oxidases including spermine oxidase and monoamine oxidase. As a whole, overexpression of the group of different genes seems to indicate that an oxidative burst could take place during cisplatin administration. We therefore searched for evidences of superoxide anion and hydrogen peroxide by means of electron paramagnetic resonance (EPR) spectroscopy and flow cytometry, but failed to detect them. On the other hand, we found an increase of malondialdehyde (MDA) synthesis and protein carbonylation products, indicating the occurence of lipid peroxidative degradation. When we tested the effectiveness of butylated hydroxytoluene (BHT), dithiothreitol (DTT) and N-acetylcysteine (N-Ac) as cytoprotectants, all of them reduced protein carbonylation to control levels and significantly protected OC-k3 from CDDP-induced cell death, with an higher protection when using the lipophylic antioxidant BHT. The same antioxidants prevented also the onset of protein carbonylation, which extent was decreased to basal levels. These data indicate that CDDP is able to stimulate gene expression up to 12 h after the beginning of the treatment. This increase in gene transcription involves a large number of genes potentially able to increase the level of cell ROS. Consistently, cells survival is improved by cotreatment with antioxidants, in particular lipophilics.

A TEOAE screening protocol based on linear click stimuli: performance and scoring criteria.

In order to improve the quality of current TEOAE recording methodologies, we have conducted a comparison of TEOAE neonatal recordings acquired with linear protocols using click stimuli of 68 dB SPL and non-linear protocols using the ILO default stimulus values. From a theoretical standpoint it was expected that the linear recordings would generate responses characterized by higher S/N ratios due to the fact that the stimulus sequence contains four clicks of the same intensity and polarity. The project included recordings from 1,416 neonatal ears (age 48 h). The TEOAE data were compared in terms of correlation, response amplitude, noise, corrected response and S/N ratio in the 1.0-, 2.0-, 3.0-, 4.0- and 5.0-kHz bands, using a paired t-test criterion. We found that windowed (4-14 ms) responses evoked by a linear TEOAE protocol generated superior S/N estimates in the 2.0-, 3.0-, 4.0- and 5.0-kHz TEOAE bands, in addition to superior correlation estimates, and demonstrated lower levels of noise. Clear-cut scoring criteria were established for the S/N ratios at 2.0, 3.0 and 4.0 kHz, by constructing one-sided distribution-free tolerance boundaries.

Efficient otoacoustic emission protocols employed in a hospital-based neonatal screening program

Within the context of a hospital-based newborn hearing screening program, we have studied the application of two OAE protocols (TEOAE and DPOAE) on a group of 250 well babies. The main goal of this study was to evaluate the performance of DPOAE protocol in a relatively large population sample. using a preset number of five tested frequencies, in comparison with a default TEOAE screening protocol. The data were collected on the second day of life and during spontaneous sleep. The TEOAE recordings were acquired with linear protocols using click stimuli of 70-75 dB SPL and were used as indicators of normal cochlear function. The cubic distortion product DPOAE responses were evoked by an asymmetrical 75-65 dB SPL protocol, with a frequency ratio of 1.22. Five frequencies (referring to F2) were tested at 1.5, 2.0, 3.0, 4.0 and 5.0 kHz. The data from the DPOAE responses show a similar pass rate (similarity = 0.98) to the linear TEOAE protocol. The data presented suggest that a DPOAE cochlear evaluation, at 5 pre-selected frequencies, has clinical potential.Within the context of a hospital-based newborn hearing screening program, we have studied the application of two OAE protocols (TEOAE and DPOAE) on a group of 250 well babies. The main goal of this study was to evaluate the performance of DPOAE protocol in a relatively large population sample, using a preset number of five tested frequencies, in comparison with a default TEOAE screening protocol. The data were collected on the second day of life and during spontaneous sleep. The TEOAE recordings were acquired with linear protocols using click stimuli of 70-75 dB SPL and were used as indicators of normal cochlear function. The cubic distortion product DPOAE responses were evoked by an asymmetrical 75-65 dB SPL protocol, with a frequency ratio of 1.22. Five frequencies (referring to F2) were tested at 1.5, 2.0, 3.0, 4.0 and 5.0 kHz. The data from the DPOAE responses show a similar pass rate (similarity=0.98) to the linear TEOAE protocol. The data presented suggest that a DPOAE cochlear evaluation, at 5 pre-selected frequencies, has clinical potential.