Cathepsins K, L, and S are cysteine proteases upregulated in cancer and proteolyze extracellular matrix to facilitate metastasis, but difficulty distinguishing specific cathepsin activity in complex tissue extracts confounds scientific studies and employing them for use in clinical diagnoses. Here, we have developed multiplex cathepsin zymography to profile cathepsins K, L, and S activity in 10 μg human breast, lung, and cervical tumors by exploiting unique electrophoretic mobility and renaturation properties.Frozen breast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same human patients were obtained , minced and homogenized prior to loading for cathepsin gelatin zymography to determine enzymatic activity.Cleared bands of cathepsin activity were identified and validated in tumor extracts and detected organ- and stage-specific differences in activity. Cathepsin K was unique compared to cathepsins L and S. It was significantly higher for all cancers even at the earliest stage tested , and stage II for breast; n = 6, p < .0001). Interestingly, cervical and breast tumor cathepsin activity was highest at the earliest stage we tested, stages I and II, respectively, and then were significantly lower at the latest stages tested  , but lung cathepsin activity increased from one stage to the next . Using cathepsin K as a diagnostic biomarker for breast cancer detected with multiplex zymography, yielded 100% sensitivity and specificity for 20 breast tissue samples tested  in part due to the consistent absence of cathepsin K in normal breast tissue across all patients.To summarize, this sensitive assay provides quantitative outputs of cathepsins K, L, and S activities from mere micrograms of tissue and has potential use as a supplement to histological methods of clinical diagnoses of biopsied human tissue. Tumor growth, migration, invasion and metastasis involves proteolytic activity, and the cathepsin family of cysteine proteases are proteases that have been implicated in each of these mechanisms, particularly cathepsins B, K, L, and S ,2. CatheCathepsins K, L, and S are elastinolytic and collagenolytic cysteine proteases that share greater than 60% sequence homology . It was Cathepsin K has been particularly elusive in measuring its activity in cancer specimens. A number of studies have implicated cathepsin K expression in cancer progression and metastasis using cathepsin K inhibitors ,20, mRNAHere, we describe multiplex cathepsin zymography, a technique that we recently developed that was capable of detecting cathepsin K activity down to femtomolar levels of recombinant enzyme and in macrophage derived osteoclasts . CathepsBreast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same patients were purchased from Protein Technologies Inc., San Diego, CA which is facilitated by Integrated Laboratory Services-Biotech (ILSbio). The original tumor and normal tissue specimens were collected from multiple hospitals. Tissue specimens were collected during the surgery process and immediately snap frozen with liquid nitrogen. ILSbio collected specimens under local Institutional Review Board approved protocols, ensuring each sample had patient consent for research purposes. 28 breast tissues, 23 lung tissues, and 23 cervix tissues were obtained Table . Tumor sCathepsin zymography was performed as described previously . BrieflyMMP zymography was similar except the enzymes were renatured in 2.5% Triton-X and incubated in 50 mM Tris-HCl pH 7.4, 10 mM calcium chloride, 50 mM sodium chloride, 0.05% Triton-X assay buffer overnight. Gels were imaged using an Imagequant 4010 . Images were inverted in Adobe Photoshop and densitometry was performed using Scion Image. Representative zymograms shown here have had the levels adjusted for the entire gel image to improve print viewing clarity. All human recombinant cathepsins were from Enzo Life Sciences . Human cathepsins K and S were expressed in insect cells, human cathepsin V was expressed in NSO cells, and human cathepsin L was isolated from liver.SDS-PAGE was performed, and protein was transferred to a nitrocellulose membrane (Bio-Rad) then probed with monoclonal anti-cathepsin K antibody clone 182-12G5  or anti-cathepsin L, or S antibodies . Secondary donkey anti-mouse or anti-goat antibodies conjugated to an infrared fluorophore  were used to image protein with a Li-Cor Odyssey scanner .Results are shown as mean ± SEM of normal and tumor groups. Student's unpaired t-test was used to evaluate statistical significance between two result groups. Values of p < 0.05 were considered statistically significant. Sensitivity, specificity, and likelihood ratio of the corresponding protease biomarker were calculated across a range of threshold values with Matlab (Mathworks). To determine the optimal threshold value that would maximize sensitivity and specificity, we input the range of values from zero to the larger value of either the maximum protease value measured in normal specimens or the minimum value measured in the cancer specimens. Threshold window index was calculated according to the following formula:Mature cathepsins K, L, and S were loaded for cathepsin zymography and parallel samples were loaded for Western blotting to first determine if the zymographically active bands of cathepsins K, L, and S would appear at different electrophoretic migration distances. Different amounts of each cathepsin were loaded to produce clear bands in the zymogram as they have different limits of detection by the zymography assay (data not shown). Cathepsins K, L, and S  all appeared as zymographically active bands at distinct molecular weights Figure . MigratiOnce it was determined that cathepsins K, L, and S could be detected with cathepsin zymography, we tested the hypothesis that cathepsin K activity would be significantly increased in breast cancer tissue compared to normal tissue, and that zymography would detect these differences. Equal amounts of breast tissue protein 10 μg) were loaded for cathepsin zymography and quantified by densitometry Figure . In thes0 μg wereMatrix metalloproteinases (MMPs) are another family of proteases that are metal dependent endopeptidases implicated in cancer development and metastasis ,30. MMP-We next wanted to determine any stage specific differences in breast cancer cathepsin activity using this cathepsin zymography assay. At least five different specimens each of stages II, III, and IV breast tumor tissue  and normal tissues were obtained and loaded for cathepsin zymography. Stage I and premalignant breast tissue samples were unavailable to us. Cathepsin activity peaked at stage II and declined through stages III and IV Figure . It is iPatient-to-patient variation in cathepsin K and L activity was assessed to determine if a threshold value of cathepsin K activity could be set that, once crossed would indicate a positive cancer specimen, Figure . AbsolutSensitivity and specificity analyses were performed to quantify the probability of a sample being correctly or incorrectly diagnosed by zymography for cathepsins K and L. Likelihood ratios were calculated to select the maximum sensitivity and specificity for each protease tested, and the ranges of values over which the likelihood ratio is maximized are highlighted by the yellow box Figure . CathepsWith successful detection of mature cathepsins K, L, and S in human breast cancer tissue, other types of tumors were investigated to establish broader utility of this assay as a screen for multiple cathepsins in one tissue specimen. Cathepsin K had been previously identified immunohistochemically in lung tumor specimens ,32, but Multiple proteases have been shown to be related to cervical cancer development ,34, but Cervical tumor specimens' cathepsin K activity displayed a wide range of patient-to-patient variability, as seen in the box-whisker plot, and, as a result, comparisons of all normal samples to all tumor samples was not statistically significant. However, there were significant differences determined between normal cervical tissue and stage I and stage II cervical cancer tissue, but not that of stage III Figure . To remoTo compare cathepsins K, L, and S activity across all three tissues tested and observe any differences in normal baseline signatures as well as cancer-mediated increases, 10 μg of protein from each organ, normal and tumor, were loaded into one zymogram Figure . Lung baMultiplex zymography's utility as a supplemental screening tool of pathological specimens was effectively shown here to profile cathepsin K, L, and S activities in breast, lung, and cervical tissue at three different stages of tumor progression. This matrix of information was captured by this one assay after clinical grading of the biopsied tissue indicating that quantitative comparisons with cathepsin zymography can supplement the gold standard histological methods of determining whether biopsied tissue is cancerous or not.Differences in organ and tissue structure or predominant extracellular matrix (ECM) components may be responsible for the differences in cathepsin K, L, and S activity profiles between breast, lung, and cervical tissue and changes to these profiles as the cancer stage increased. Ductal carcinoma breast cancers arise in the inner layer of mammary duct in the columnar epithelium that lines it, and are surrounded by lobes, stromata, and adipose tissues. Squamous cell carcinoma of the cervix starts in the epithelium of cervix and invades into the underlying stroma. Lung tumors, however, that mainly arise in the bronchi, are surrounded by hyaline cartilage, a tough connective tissue made of high density of collagen II and is also in a much more dynamic environment as the diaphragm contracts and relaxes during breathing. ECM proteins , mechaniOf the three types of cancer and the three cathepsins studied, cathepsin K in breast tissue was especially unique in that its activity was binary: off in normal tissue and on in cancerous tissue. Cathepsin K in breast tissue had the lowest variability and consistently low baseline for cathepsin K activity in the normal tissue, compared to breast, lung, and cervical cancer tissue Figure . This suHigher patient-to-patient variability of cathepsin levels within lung and cervical cancer specimens tested here may be due to the source of the tumorigenicity for each organ. National Cancer Institute reports that smoking is the leading cause of lung cancer deaths: 90 percent for men and 80 percent for women (NCI 2010), and differences in smoking habits and tobacco delivery methods may be the cause of the variability detected by this assay. Despite the variability within a stage of lung cancer, there was still a statistically significant increase in cathepsin activity compared to normal. Again, cathepsin K activity stood out as being significantly upregulated even at stage I of lung cancer, where cathepsin L and S did not reach this point until stage II Figure . Unique Human papilloma virus (HPV) infection is a leading cause of cervical cancer  and has Pap smears are routinely performed to screen for cervical cancer. Small samples of cervical tissue are biopsied and clinical and pathological grading of the histology is performed to observe any abnormal cells in the samples. Given the results shown here with a 10-fold increase in cathepsin K activity detected from 10 μg of tissue protein, cathepsin zymography may serve as a supplemental biomarker to aid the assessment of inconclusive Pap smear results. Again, more clinical samples will need to be tested to verify its utility, but cathepsin K also presented an on/off activity in cancer vs. normal cervical tissue, similarly to breast tissue.Cervical and breast cancer cathepsin activity peaked at the earliest stage we tested, stages I and II, respectively, and then were significantly less at the latest stage tested. This non-intuitive change in cathepsin activity in the primary tumor has not previously been shown. Tumor cell heterogeneity may provide one possible explanation. Our hypothesis is that the more metastatically inclined cells in the primary tumor are producing more cathepsin proteases to facilitate departure from the primary tumor site; when they leave the primary tumor, the source of the proteolytic activity leaves as well. This finding may guide improved stage specific treatments for tumors and indicate more rigorous protease inhibition strategies at primary tumors to block the earliest steps of metastasis.It is important to note this zymography assay samples the entire tumor, not just the tumor cells. Therefore, any tumor associated macrophages, blood vessels, white blood cells, or any other infiltrating cells with cathepsin activity will be captured in that tissue extract. Aggressive tumor cells are able to recruit the surrounding stromal cells to enhance tumor growth. Tumor associated macrophages expressing cathepsins were shown to organize around the tumor edge at later stages in a pancreatic cancer animal model , indicatOverall, the application of multiplex cathepsin zymography to breast, lung, and cervical cancer specimens have highlighted the unique upregulation of cathepsin K in all three of these cancers tested and even in the earliest stages measured. Altogether, this elevates cathepsin K potential to be a cancer biomarker for breast, lung, and cervical cancer, but more broadly, highlights its potential to be a biomarker for other types of cancer that have not previously been investigated. Histological studies benefit from quantitative, visual confirmation offered by cathepsin zymography using just a small piece of the biopsied tissue. As an added benefit to researchers, this assay does not require antibodies, which expands its application to other species, including the numerous mouse models of cancer that have been developed. Lack of antibodies also significantly reduces cost compared to immunobased methods such as ELISA, Western blotting, and immunohistochemistry, as well as minimizing concerns of nonspecific antibody binding and pro-cathepsin detection interference. Employing such multiplex technologies, that can screen samples inexpensively, will provide a broader net to catch new biomarkers and etiological agents to direct investigation into previously untested mechanisms and inhibitory targets.The authors declare that they have no competing interests.BC participated in the design of the study, conducted experiments, performed statistical analysis, and helped draft the manuscript. MOP conceived of the study and participated in its design and coordination, conducted experiments, and helped to draft the manuscript. All authors read and approved the final manuscript.Diagnostic performance of MMP-2 and MMP-9 for breast cancer. Sensitivity (blue line), specificity (red line), and likelihood ratio (dotted black line) were calculated and plotted over a range of values to identify an optimal threshold value for MMP-2 and MMP-9 that would distinguish normal samples from tumor samples. Yellow boxes outline the region of maximal likelihood ratio.Click here for file


Background. This study investigated whether cortical auditory evoked potentials (CAEPs) could reliably be recorded and interpreted using clinical testing equipment, to assess the effects of hearing aid technology on the CAEP. Methods. Fifteen normal hearing (NH) and five hearing impaired (HI) children were included in the study. NH children were tested unaided; HI children were tested while wearing hearing aids. CAEPs were evoked with tone bursts presented at a suprathreshold level. Presence/absence of CAEPs was established based on agreement between two independent raters. Results. Present waveforms were interpreted for most NH listeners and all HI listeners, when stimuli were measured to be at an audible level. The younger NH children were found to have significantly different waveform morphology, compared to the older children, with grand averaged waveforms differing in the later part of the time window (the N2 response). Results suggest that in some children, frequency compression hearing aid processing improved audibility of specific frequencies, leading to increased rates of detectable cortical responses in HI children. Conclusions. These findings provide support for the use of CAEPs in measuring hearing aid benefit. Further research is needed to validate aided results across a larger group of HI participants and with speech-based stimuli. A growing body of literature exists on the use of cortical auditory evoked potentials (CAEPs) in assessing neural activity in NH and HI listeners. Such measures reflect the sum of synchronous, time-locked neural activity detected at the level of the central auditory system, related to the strength (amplitude) and timing (latency) of a response , 2. For When sound is processed through a hearing aid, it is necessary to understand what the hearing aid is doing to the signal. For this reason, recent research in this area has focused on amplification-related modifications to the CAEP stimulus, related to factors such as poor signal-to-noise ratio (SNR) and/or rise time –13; bothOne example of a change in hearing aid signal processing is the application of frequency lowering. Studies suggest that frequency lowering hearing aid technology may benefit adults and children with high-frequency hearing loss  of a hearing aid with an audioshoe connector in aided testing conditions. The purpose of this study was to investigate the following: (1) whether CAEPs could be reliably recorded and interpreted using Bio-logic Navigator Pro testing equipment in NH and HI children, and (2) whether CAEPs elicited by high-frequency tone burst stimuli reflected the change in high frequency audibility due to use of NLFC hearing aid technology. Participants included 15 NH children  and 5 HI children with high-frequency hearing loss . Children in this age range were chosen for this pilot study as they are likely to understand and follow instructions during testing  and their CAEPs may be considered representative of a maturing system . NH chilThe eligibility criteria for the NH participants included passing a hearing screen at 15 dB HL. For the HI participants, hearing thresholds were measured by coupling the insert earphones to each participant's personal earmolds. Eligibility criteria included bilateral sensorineural hearing impairment, sloping to at least a moderately severe high-frequency pure-tone average (HF-PTA) hearing level averaged across 2, 3, and 4 kHz. Hearing threshold data is displayed for each case in the Results section. Participants were required to be full-time users of digital behind-the-ear (BTE) hearing aids prior to entering the study and to maintain full time use of the study hearing aids prior to CAEP measurement. Four of the participants were binaural hearing aid wearers with symmetrical high-frequency hearing loss within 10 dB, based on HF-PTA. One participant with an asymmetrical hearing loss was monaurally aided in the better ear (Case 5); a hearing aid trial on the ear with a greater level of impairment  was not successful. Hearing thresholds were measured at the beginning and end of the study. All participants demonstrated hearing levels within 10 dB of baseline over the course of the study. The presence of cochlear dead regions was assessed using the TEN test in dB HL .Tone bursts at 2 and 4 kHz were generated from the Bio-logic Navigator Pro (v7.0.0). Using stimulus parameter options available within the Bio-logic software, cycle numbers were held constant across stimuli with a rise/fall Blackman ramp of 20 cycles each and a plateau of 80 cycles. This produced a 40 msec plateau and 10 msec rise/fall time for the 2 kHz tone burst and a 20 msec plateau and 5 msec rise/fall time for the 4 kHz tone burst. Testing was carried out in sessions no longer than two hours. One session was required for NH participants and two were required for HI participants. Breaks were given when requested. Participants were seated watching a muted movie of their choice with active subtitles and were instructed to ignore the stimuli played , 29. TheFor the HI participants, aided monaural testing was carried out for the better listening ear, according to pure-tone average (PTA). Stimuli were routed directly from the Navigator Pro (and through an audio isolation transformer) to the DAI of the hearing aid with an audioshoe connector coupled to a study worn hearing aid, using a custom cable . This DADevice fitting followed protocols from the Desired Sensation Level (DSL) method version 5.0 , 32 as iAided CAEP testing was completed on two different testing sessions: the first with NLFC enabled (treatment condition) and the second with NLFC disabled (no-NLFC). On average, the NFLC testing session was completed after 15 weeks of acclimatization to NLFC processing (range: 14 to 17 weeks). The no-NLFC testing session was completed 4 weeks after finishing the treatment condition of the study (range: 1 to 7 weeks). Participants were naïve to all details pertaining to the study design. Such details, along with the individualized results, were disclosed to the participants upon completion of the study. The potential for hearing aid-induced delay affecting latency values in HI testing conditions was measured using an anechoic box (B&K 4232). Stimuli were presented by playing 2 and 4 kHz tone burst stimuli from SpectraPlus software via a study hearing aid connected to the coupler. The output of the coupler and reference channels of the recordings were compared to each other using a 75 dB SPL input level to estimate the presence of any hearing aid-induced delay. The difference in the onset of stimuli in each recording channel was calculated to be 6.7 msec, on average, across stimuli and testing conditions. Delay values ranged from 6.3 to 7 msec. Due to the insignificance of the calculated delay values compared to the latency of CAEPs, no corrections were applied for the purpose of comparing HI and NH CAEP data.A suprathreshold presentation level was determined by generating a tone burst stimulus from the Navigator Pro at a testing dial level of 70 dB in the anechoic test box. The output was measured via broadband insert earphones connected to an HA-2 (25 mm tubing) 2cc coupler and microphone (B&K 4192). SpectraPlus software was used to capture the output RMS of the coupler in dB SPL across the tone burst plateau. The chosen dial level produced presentation levels of 69 and 71 dB SPL (re: 2cc coupler) for the 2 and 4 kHz tone burst stimuli, respectively. The same dial level was used for aided testing with HI participants. Additional electroacoustic verification measures of aided stimuli were completed using the same set-up as described previously. Electroacoustic measures allowed individualized estimation of audibility of the tone bursts for each of the hearing aid fittings and testing conditions. Navigator Pro tone bursts generated at the chosen dial level were routed through the study hearing aid set-up, programmed with individualized fittings. Overall rms of each tone burst  was measured for individual fittings with and without NLFC for each stimulus. Since these measures were made in a 2cc coupler, audiometric thresholds were transformed to SPL in a 2cc coupler using individualized ear canal transforms , and theμV. Two averages of 100 sweeps each were obtained for each stimulus condition. Due to study-specific modifications to the traditional testing parameters used in the Bio-logic software, including epoch time/stimulus rate, it was not possible to include a calculation of residual noise level as a stop criterion.An ipsilateral recording  was obtained using the Navigator Pro. Tone bursts were presented at the rate of 0.5 stimuli/sec. This interstimulusinterval was the same as that used to acoustically record in the aided condition. Each recorded electroencephalogram (EEG) sweep included 100 msec of prestimulus baseline (relative to tone burst onset) and 966 msec of post stimulus activity. EEG was amplified 50000 times and digitized at the rate of 480.03 Hz. Responses were bandpass filtered between 0.1 and 100 Hz online. The artifact rejection threshold was set to ±100 Data extraction from the Bio-logic Software was limited to averaged data; therefore, previously reported statistical techniques  could noResearch on the maturational effects associated with the CAEP response suggests that changes to the waveform morphology associated with the P1-N1-P2 complex can be observed between the ages of 6 and 18 years . TherefoP < 0.001), 95% CI , with a standard error of zero. A Kappa value of one implies perfect agreement between the two raters.An interrater reliability analysis using the Kappa statistic was performed using SPSS software to examine consistency among the two raters. Analyses were performed including waveforms from all participants in both conditions. The interrater reliability analysis for the examiners, across all stimuli and conditions, was found to be Kappa = 1 (The intraclass class correlation coefficient (ICC) has recently been used to quantify similarity between two waveforms , 39. VisData collected with NH listeners is displayed at the group level according to grand mean waveforms per stimulus , as wellμV in the 4th bin (corresponding to latency region of 150−199 ms) to as large as 3.28 μV in the 6th bin (corresponding to latency region of 250–299 ms). On average, the 6th bin measured 0.38 μV in the older group compared to −2.9 μV in the younger group. The amplitude of the CAEP waveforms elicited to the 4 kHz tone burst varied as little as 0.003 μV in the 5th bin (corresponding to latency region of 200−249 ms) to as large as 3.03 μV in the 6th bin (corresponding to latency region of 250–299 ms). On average, the 6th bin measured 0.44 μV in the older group compared to −2.59 μV in the younger group. A significant interaction is reported between age group and time interval for the 2 kHz tone burst  = 3.73), P < 0.05) and for the 4 kHz tone burst  = 4.11), P < 0.01). Results suggest a need to separate mean CAEPs by age group when comparing NH listeners to their HI counterparts in the present study  in 15 NH (unaided) and 5 HI (aided) child listeners (ages 11–18 years). Firstly, this study investigated whether CAEPs could be reliably recorded and interpreted with NH and HI listeners using Bio-logic Navigator Pro testing equipment. Secondly, a case-series approach was used to examine the effects of NLFC hearing aid technology on CAEPs elicited by tone bursts. To facilitate evoked recordings, modifications to the standard testing parameters/equipment were made. In the aided testing conditions, equipment modifications allowed stimuli to be delivered directly to the hearing aid using a DAI hearing aid connector (coupled to an audioshoe). This eliminated the need to control for factors that could have affected the accuracy and reliability of sound-field presentation of stimuli. Due to software limitations, only averaged waveforms could be exported and analyzed offline. Waveforms were subjectively interpreted by two experienced examiners using a rating protocol. Literature suggests that that statistical detection of CAEPs is consistent with those of experienced examiners , 42. ThiWaveforms recorded with the NH listeners were found to differ across the age range included in this study. Displayed grand averaged waveforms, separated according to age, suggest that the main source of variability lies within the 250–350 msec range of the response window, with larger N2 amplitudes observed with the younger NH group. These findings are consistent with those reported for a group of normally hearing children listening to click trains: changes in the latency and amplitude values associated with the N2 response were observed up to approximately age 17 . ResultsConsidering the HI listeners, a unique pattern of results was observed across the different cases. The differences observed in waveform morphology may relate to factors including the age range of the participants included in the study  and differences in hearing loss configuration across participants and/or chosen hearing aid settings. In summary, CAEPs were sensitive to changes in audibility across NLFC and no-NLFC hearing aid conditions. Responses appear to be larger, on average, for the 2 kHz tone burst, compared to the 4 kHz tone burst. This is consistent with findings of earlier studies that report an inverse relationship of CAEP amplitude and frequency of tone burst stimuli. The peak-to-peak amplitude was found to decrease as the frequency of the tone bursts increased , 47. When comparing NH grand mean waveforms (separated by age group) to the individual averaged waveforms for the HI cases, aided CAEPs tend to be larger in amplitude than the unaided CAEPs measured with the NH group. However, this appears to be the only uniform pattern of results between CAEPs measured with NH and HI participants. Although the ages of the participants were relatively well matched across NH and HI children, the groups were tested with different transducers (broadband insert earphones versus direct audio input to a well-fitted hearing aid). Also, the sensation level of the stimuli was greater for the NH participants than for the HI participants. The HI children in this study had substantial hearing losses, likely accompanied by reduced dynamic ranges of hearing. If we had presented at matched sensation levels across the groups, the stimuli would have been either too loud for the HI participants or very soft for the NH participants. Instead, we presented at middynamic range for all listeners, which in turn results in high sensation levels for NH and low sensation levels for HI participants, respectively. Future studies could potentially consider presenting stimuli at equal loudness by incorporating a loudness model that accounts for the effects of sensorineural hearing loss . This mVariable CAEP waveform morphology can be observed across the different HI cases when considering gross amplitude and latency differences. For example, present waveforms in Case 3 are larger in amplitude when compared to all other cases. This is also the only case where a suspected cochlear dead region was measured. It is possible that the large response measured with the 2 kHz tone burst reflects overrepresentation of neurons in the cortical regions bordering the cochlear dead region. These findings are closely aligned with those reported in animal model research, describing the use of auditory evoked potential measures to quantify cortical over-representation  corresponding to the frequency region at the cut-off slope of the audiogram in cats , 50. FurAll HI participants were long-time hearing aid users who had received a period of time to acclimatize to NLFC prior to beginning testing. The length of time allotted for acclimatization for the purpose of this study is consistent with that reported in the literature  investigating acclimatization effects in aided speech perception , 52. Pregross changes in high-frequency audibility, the resulting effects on the CAEP response could be observed as either present or absent. However, further research is needed to assess the effects of hearing aid fine tuning , or other aspects of hearing aid signal processing, on CAEPs. In addition, more research is needed to validate aided findings reported in this study across a larger group of HI participants including infants and with speech-based stimuli.In summary, this study demonstrated that CAEPs can be recorded with clinical testing equipment in NH and HI children. In the aided testing condition, tone burst stimuli were directly presented to the hearing aid via a DAI connector. Repeatable present waveforms were measured in most participants, although were missing in a small number of NH listeners. Present waveforms in the HI children may have been associated with higher sensation levels. The younger NH children were found to have significantly different responses than the older NH children, with grand averaged waveforms differing mainly between 250 and 350 msec in the response window (the N2 response). For most of the HI cases included in this study, frequency compression hearing aid technology improved audibility of the 4 kHz tone burst; this translated into improved detection of CAEP responses. These findings suggest that the CAEP may be sensitive to the effects of frequency compression signal processing and that frequency compression may have augmented audibility of high-frequency tone bursts on a case-by-case basis. This contribution to the literature provides insight into possible strengths (sensitivity to changes within the aided condition) and limitations  of CAEP. As this study evaluated


P301S) mice develop tau filamentous inclusions and axonopathy in retinal ganglion neurons (RGCs), in the absence of neuronal loss or alterations in the outer retina. Moreover, we showed that tauP301S transgenic retinal explants do not respond to neurotrophic stimuli in vitro. Here, we investigated the impact of tau pathology on RGC physiology in living animals and neurotrophin signaling pathways in vivo. In anesthetized 5-month old wild type (WT) and tauP301S mice, we measured RGCs activity using pattern electroretinogram (pERG), which selectively detects RGC response upon pattern light stimuli exposure. In transgenic tauP301S mice the amplitude of both P1 positive and N2 negative components of pERG at saturating contrast and spatial frequency was significantly smaller than WT values. Furthermore, retinal acuity was significantly reduced in tauP301S mice. Using uniform flickers of light (flash ERG), we measured the activity of the outer retina and found that outer retina response was preserved in tauP301S mice. Neurotrophins, and especially brain-derived neurotrophic factor (BDNF), are important modulators of neuronal survival and function in the brain and in the visual system. We therefore investigated the BDNF signaling pathway and found that BDNF signalling was altered in tauP301S transgenic retinas. Our results indicate that, in the tauP301S mouse, tau pathology specifically impairs the activity of RGCs, without affecting the outer retina function and is associated with BDNF signalling alterations. Given the role of BDNF in synaptic plasticity, these data suggest that mild levels of tau pathology are sufficient to trigger significant neuronal dysfunction possibly through alteration of neurotrophic signalling. Funded by a grant of Compagnia di San Paolo awarded to LG.Intracellular inclusions made of microtubule-associated tau protein are a defining pathological hallmark of tauopathies, which include Alzheimer disease and familial frontotemporal dementia and parkinsonism linked to chromosome 17. Altered levels of tau protein have been detected in the retina and optic nerve of patients with glaucoma, suggesting that retina degeneration and tauopathies share similar pathogenic mechanisms. We have recently demonstrated that P301S mutant human tau (tau


Recent investigations demonstrated many genetic contributions to the development of human age-related hearing impairment (ARHI), however, reports of factors associated with a reduction in the ARHI risk are rare. Folate metabolism is essential for cellular functioning. Despite the extensive investigations regarding the roles of folate metabolism related gene polymorphisms in the pathophysiology of complex diseases, such as cancer, cardio-cerebrovascular disease, and atherosclerosis, little is known about the association with ARHI. The aim of this study is to investigate the effects of the methionine synthase (MTR) A2756G and methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphisms on the risk of hearing impairment in middle-aged and elderly Japanese.Data were collected from community-dwelling Japanese adults aged 40-84 years who participated in the Longitudinal Study of Aging biennially between 1997 and 2008. We analyzed cumulative data  using generalized estimating equations.The MTHFR 677T allele was significantly associated with a reduced risk of hearing impairment only when the subjects were wild-type homozygotes for MTR A2756G. The per-T allele odds ratio of MTHFR for the risk of developing hearing impairment was 0.7609 (95% CI: 0.6178-0.9372) in the MTR AA genotype. In addition, a subgroup analysis demonstrated that the favorable effect of the MTHFR 677T allele on the risk of developing hearing impairment was independent of folate and homocysteine level, whereas plasma total homocysteine level was independently associated with an increased risk of developing hearing impairment. The interactive effect of gene polymorphisms associated with folate metabolism may modify the risk of developing hearing impairment after middle age. These results contribute to the elucidation of the causes of ARHI.The present study has found that the MTHFR 677T allele has a favorable effect on a risk of hearing impairment in the middle-aged and elderly population, only when the individuals were wild-type homozygotes for MTR A2756G. Presbycusis is one of the most common sensory impairments affecting the elderly and is a multifactorial process that involves a multitude of intrinsic and extrinsic factors. Recent investigations demonstrated many genetic contributions to human age-related hearing impairment (ARHI) -7.de novo synthesis of purines and pyrimidines, which are necessary for RNA and DNA synthesis, and provides methyl groups, which are necessary for the remethylation of homocysteine and for methylation reactions such as DNA methylation  and 0.4 mmol/L BCIP , a substrate for alkaline phosphatase, were added and colorimetry was performed. The genotypes were identified according to the absorbance signal ratio between C type-specific and T type-specific wells. Further details of MTHFR genotyping in the NILS-LSA are supplied elsewhere .2, and 1 U of rTaq DNA polymerase  in the appropriate DNA polymerase buffer. The amplification protocol consisted of an initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 2 minutes. Amplified DNA was incubated in a solution containing streptavidin-conjugated magnetic beads in the wells of a 96-well plate at room temperature. The plate was placed on a magnetic stand, and the supernatants were then collected from each well, transferred to the wells of a 96-well plate containing 0.01 mol/L NaOH, and fluorescence was then measured using a microplate reader  at excitation and emission wavelengths of 485 nm and 538 nm, respectively, for fluorescein isothiocyanate and 584 nm and 612 nm, respectively, for Texas Red.The polymorphic region of the gene was amplified using PCR and two ASPs labeled at the 5" end, either with fluorescein isothiocyanate  or with Texas Red  and a biotin-labeled common antisense primer (5'-biotin-CTACCACTTACCTTGAGAGACTCAT-3'). The reaction mixture (25 μL) contained 20 ng of DNA, 5 pmol of each primer, 0.2 mmol/L of each deoxynucleoside triphosphate, 5.0 mmol/L MgClAir-conduction pure-tone thresholds at octave intervals from 0.5 kHz to 8 kHz were measured in a sound-proof booth by trained examiners using a standardized protocol and a diagnostic audiometer . For analyses of supraliminal levels, thresholds in excess of the predetermined output level according to the JIS  calibration plus an additional 5 dB were treated as levels. The average hearing threshold level (AHT) of the better ear at frequencies of 0.5, 1, 2, and 4 kHz was used as an index of hearing status among genotypes. Hearing impairment was defined as an AHT greater than 25 dB according to World Health Organization grades .Venous blood samples were collected with the consent of the participants for genetic and other blood analyses. Blood samples were collected early in the morning after at least 12 hours of fasting. Plasma total homocysteine concentration was determined using high-performance liquid chromatography and fluorimetric detection . The ser2).Most of the moderator variables for analyses  were self-reported. Histories of occupational noise exposure and lifetime smoking were categorized as never vs. ever. Individual medical histories were obtained using questionnaires. Each of the cited moderators was treated as a binary variable (presence vs. absence). Body mass index (BMI) was calculated as weight (kg) divided by height . Univariate analyses of categorical variables according to hearing impairment status were performed using the chi-square test and baseline data. The chi-square test was also conducted to assess the independency of genotype distribution of the two polymorphisms in the baseline data. Cumulative data were analyzed using generalized estimating equations (GEEs), which take into account the dependency of repeated observations within subjects. GEE models were fitted using the GENMOD procedure of SAS. The GENMOD procedure fits generalized linear models. The correlation structure was specified as autoregressive.Genotypes were coded as follows: wild-type homozygotes, AA for MTR and CC for MTHFR, heterozygotes, AG for MTR and CT for MTHFR, and mutant homozygotes, GG for MTR and TT for MTHFR. In an exploratory analysis to assess the effect of MTR A2756G and MTHFR C677T on hearing impairment, MTR, MTHFR, and the MTHFR by MTR interaction were set as independent variables using the GENMOD procedure. Then, based on the results of the exploratory analysis, odds ratios (ORs) for the additive genetic models of MTHFR polymorphisms according to MTR genotypes with the risk of hearing impairment were analyzed with adjustment for age, sex, smoking status, BMI, and histories of occupational noise exposure, heart disease, hypertension, and diabetes. The additive genetic model, which is the prevailing analytical model in genetic epidemiology, assumes that there is a linear gradient in risk with increasing numbers of mutated alleles. The per-allele OR for the risk of developing hearing impairment was determined using the additive genetic model.Data from only 1,146 of the 1,426 baseline participants could be used for a subgroup analysis in which blood folate and homocysteine levels were included as variables in the statistical model because blood folate and homocysteine levels were measured during first-wave examinations. Comparison of serum folate and plasma total homocysteine levels according to MTHFR genotypes by MTR genotypes was conducted using a general linear model. A multiple logistic regression was then performed to obtain ORs for hearing impairment in subjects with the MTHFR C677T polymorphism by MTR genotypes as a subgroup analysis. In this analysis, age, sex, smoking status, BMI, and histories of occupational noise exposure, heart disease, hypertension, and diabetes were denoted as possible influential variables. ORs per ng/mL increase in serum concentration of folate and per nmol/mL increase in plasma total homocysteine concentration were also estimated.All values are expressed as the mean ± standard deviation unless otherwise specified. A null-hypothesis probability of < 0.05 was regarded as statistically significant.p > 0.05).The general characteristics and genotype frequencies of the baseline subjects are presented in Table p = 0.0318, 0.0022, and 0.0076, respectively). After adjustment for moderators , the main effect of MTHFR was still significant (p = 0.0230), but the main effect of MTR was not significant (p = 0.2736). The interactive effect was almost significant (p = 0.0856). The per-allele OR for the risk of developing hearing impairment according to the additive genetic model of MTHFR polymorphisms by MTR genotypes with adjustment for moderators is shown in Table The genotype frequencies of the two polymorphisms in the cumulative data are presented according to hearing impairment status in Table In a subgroup analysis, the mean level of serum folate in 1,146 subjects was 5.6 ± 2.6 ng/mL, and that of plasma total homocysteine was 11.0 ± 5.1 nmol/mL. Levels of serum folate and plasma total homocysteine according to MTR and MTHFR genotypes are shown in Table The main findings of our study were that the risk of developing hearing impairment in middle-aged and elderly subjects is influenced by an interaction between the MTR A2756G and MTHFR C677T gene polymorphisms and that the MTHFR 677T allele is associated with a reduced risk of hearing impairment, but only when the MTR A2756G genotype is a wild-type homozygote (AA).p = 0.037) and 2.6 dB (p = 0.021) lower PTA-high and PTA-low hearing thresholds, respectively, than subjects with a 677C allele when their folate status was above the median. They found that high serum folate concentrations were associated with better hearing in MTHFR 677TT homozygotes, whereas high serum folate concentrations were associated with poorer hearing in CC homozygotes. In the present study, the MTHFR variant allele conferred an advantage with respect to hearing when the MTR A2756G genotype was the wild type, which probably maintains MTR enzymatic activity at a higher level than in the variant [12 deficiency or in cases of chronic ethanol ingestion, results in the continuous conversion of 5,10-methylene-THF to 5-methyl-THF. Thus, a substantial proportion of cellular folate is converted into a metabolically unavailable form, which results in a functional folate deficiency [We recently reported that the T allele of the MTHFR C677T gene polymorphism may be associated with an increased risk of SSNHL . In the  variant . Studiesficiency . The MTRAs referred to previously, the MTHFR 677T allele has favorable and unfavorable effects on human health. The conflicting effects of the MTHFR C677T polymorphism on carcinogenesis may be resolved as follows. When the dietary supply of folate and related nutrients is sufficient, individuals carrying variant MTHFR genotypes may be at reduced risk of cancer because high intracellular levels of 5,10-methyleneTHF may prevent imbalances in the nucleotide pool during DNA synthesis, thereby ensuring that DNA replication occurs with high fidelity ,13. DNA Durga et al. explained the etiology of AHRI in terms of mitochondrial damage . It was This study has certain limitations. A major weakness of this study is that homocysteine and folate levels were available for only 22% of the cumulative data because of cost restriction. Also lab tests for vitamin B12 and/or methylmalonic acid were not performed, which are additional metabolic parameters potentially influenced by MTR enzymatic action. Current opinion is that the 677 C-to-T variant exerts its effect via an elevated homocysteine level as described as 'the homocysteine hypothesis' . On the Another issue that has created some confusion is the seemingly contradictory effects of the MTHFR 677T allele on SSNHL vs. ARHI in the NILS-LSA study population. We reported that the MTHFR 677T allele may be associated with an increased risk of SSNHL and that it may be associated with a reduced risk of hearing impairment later in life if the individuals are wild-type homozygotes for MTR A2756G. No plausible explanation for this contradiction can be offered at this stage. However, given that one of the many hypotheses on the etiology of SSNHL involves impaired perfusion and ischemic damage to the cochlea, the MTHFR 677T allele may contribute to risk of an acute vascular event in the cochlea. On the other hand, the MTHFR 677T allele has certain advantages for the better-hearing ear in homozygous MTR 2756AA individuals, which are independent of plasma total homocysteine level. Bilateral hearing impairment as defined by the World Health Organization requires the presence of a hearing impairment in the better-hearing ear. Therefore, the better-hearing ear was used in our study in order not to overestimate individuals with only one affected ear as subjects with ARHI. The contribution of the MTHFR 677T allele to the affected ear in SSNHL may differ from its contribution to the better-hearing ear in middle-aged and elderly people.Although genetic contributions to ARHI have been increasingly reported, significant associations between gene polymorphisms and a reduced risk of ARHI are rare. The results of this study may help to elucidate the complex mechanisms responsible for ARHI. This is an observational epidemiological design, which helps us to understand the factors that determine the presence or absence of disorder. Hypotheses derived from our results need to be verified by further basic and applied researches.The present study has found that the MTHFR 677T allele has a favorable effect on a risk of hearing impairment in the middle-aged and elderly population, only when the individuals were wild-type homozygotes for MTR A2756G. In addition, the favorable impact of the MTHFR 677T allele on the risk of developing hearing impairment in the MTR 2756AA genotype was independent of blood folate and homocysteine levels. These results may contribute to our understanding of the pathology of ARHI.YU carried out the statistical analyses and drafted the manuscript. HS and FA designed the study, collected the samples, and managed the overall project. YU, SS and TN implemented cooperation in conducting auditory section of the study.All authors contributed to the writing of the manuscript, read and approved a final draft.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/35/prepubSupplemental figure S1. Simplified scheme of the role of MTR and MTHFR in folate metabolism and one-carbon transfer reactions. The file contains a scheme which provides a comprehensible information regarding favorable and unfavorable effects of MTHFR 677T allele on biological activity.Click here for file


The objective of this study was to present 5 years of surgical experience, and the extended results of hearing preservation (based on 3-year follow-up), with the Med-El Vibrant Soundbridge (VSB) in which the floating mass transducer (FMT) is placed directly against the round window membrane, and the fascia is used only as covering tissue to keep it in position. A retrospective survey of surgical and audiological data was conducted to evaluate the performance and stability of patient hearing, with audiometric measurements performed over fixed time intervals up to 36 months. 21 patients, aged 19–62 years (mean 48.4), with mixed or conductive, bilateral or unilateral hearing loss were included in this study. Surgical intervention involved monaural implantation of the Med-El VSB between 2006 and 2009. The results were assessed using pure tone audiometry. In 5 years of experience with the technique, no significant complications or device extrusion were observed except for two revision surgeries requiring FMT repositioning. In the 3-year follow-up, we observed stable hearing in the implanted ear. It is concluded that direct round window stimulation without interposed fascia is an alternative for patients with hearing impairment caused by chronic otitis media and/or lack of ossicles, especially after modified radical mastoidectomy. It allows good results in a selected group of patients, although further observation on a larger population is needed to confirm long-term validity and effectiveness. The Med-El Vibrant Soundbridge (VSB) was introduced in Europe in the late 1990s as a middle ear implantable hearing device that could compensate for sensorineural hearing loss. However, it took some time, until 2006, for it to be used for conductive hearing loss treatment [Previous reports on both methods of round window membrane stimulation presented by Nakajima et al.  and ArnoPennings et al. demonstrated that when using an additional covering fascia there was no large difference between both methods in energy transferred to the cochlea. The authors also drew attention to possible future complications that could involve degradation due to scarring of the fascia between the FMT and the RW membrane. However, the current state of knowledge does not allow firm conclusions to be drawn, because a longer period of observation is required. There is little doubt that success in proper positioning of the FMT against the RW membrane is related to the surgeon’s experience.The results for improving speech intelligibility and quality of hearing have already been well documented in the literature, particularly for sensorineural hearing losses, when the FMT is positioned on the long process of the incus . In compThe aim of this study was to present 5 years of surgical experience, and extended results of hearing preservation, during 3-year follow-up of patients implanted with the Med-El VSB device in which there was direct placement of the FMT against the RW using a covering fascia over the FMT, but with nothing placed between the FMT and the RW membrane (without interposed tissue) Fig. .Fig. 1PlExactly 21 patients, 16 women and 5 men, aged 19–62 years (mean 48.4) with conductive or mixed hearing loss were included in this study; they were implanted at the Institute of Physiology and Pathology of Hearing in Warsaw between 2006 and 2009. Of the 21, 6 were diagnosed with unilateral hearing loss. Demographic data are presented in Table All patients were unilaterally implanted with the Med-El VSB device with the FMT placed directly in contact with the RW membrane (without interposed tissue). Only a few patients used hearing aids prior to the operation since in the majority of cases it was contraindicated due to recurring ear effusions. For at least 1 year before implantation, there was no significant progress in the patients’ hearing losses. None of them complained of tinnitus and/or vertigo before the surgery. Patients had undergone surgical treatment—radical or radical modified mastoidectomy in one or both ears—before they were selected for implantation Table . They alThe patients were all assessed preoperatively and postoperatively over fixed time intervals to obtain hearing thresholds for both AC and BC at standard frequencies. The intervals were 0–3 months pre-op (pre); 0–1 month ; 2–3 months ; 4–6 months ; 7–12 months ; 13–24 months ; and 25–36 months . Processors were fitted with a DSL I/O formula based on the manufacturer’s recommendations. BC thresholds were used as input data to the calculation of electroacoustic parameters. In cases where there were no acceptable calculated DSL targets, further adjustment was performed on the basis of the subjective preferences of the patient.Exactly 21 patients were selected for VSB implantation with the FMT placed directly in contact with the RW (without interposed tissue); surgery was done at the International Center of Hearing and Speech of the Institute of Physiology and Pathology of Hearing, Warsaw.In all patients, an approach through the external ear canal was performed to assess the condition of the middle ear and to visualize the RW niche. In seven patients, the RW niche was occluded by adhesions as a result of chronic otitis and sometimes multiple previous surgeries; in these cases, the adhesions and scar tissue were carefully removed without damaging the RW membrane until the RW could be properly visualized. In all cases, to properly visualize the RW, the bony lip was removed with a small-diameter diamond burr. The drilling was done intermittently at slow speed and with irrigation, always avoiding contact with the RW membrane, because direct physical contact from a working drill can cause sensorineural hearing loss. If possible, only the internal part of the bony lip was removed, leaving the bony roof to form a kind of a well for the FMT. If this was not possible, the whole superior lip was completely drilled away to visualize the RW membrane. At this stage, the template was placed onto the RW, and a postauricular groove was cut. The epidermis lining the postoperative cavity was then carefully detached from the bony bed of the temporal bone to receive the internal part of the implant. A second placement check with the template was done before the FMT was placed in position. We recommend that the FMT is fixed from behind and inferiorly using a piece of fascia and fibrin glue. Next, two or three layers of larger pieces of fascia are used as an additional cover of FMT and the RW, preventing the transducer from moving. After fixing all layers to the surrounding bony surfaces with glue, everything is covered with the earlier detached parts of the epidermis taken from the external ear canal or the postoperative cavity.The final surgical steps involve placement of the internal capsule of the implant in the earlier prepared bony bed. For the next 7–8 days, the FMT and surrounding structures are covered by a foil dressing.The results of AC and BC hearing in the ear selected for surgery and in the opposite ear before implantation of the FMT are shown in Fig. p = 0.043) and 4,000 Hz (p = 0.015), and between interval II vs. pre for 4,000 Hz (p = 0.03). Comparison of hearing for BC thresholds before and 36 months after direct placement of FMT against the RW showed no statistically significant differences for all tested frequencies . Statistically significant changes were also observed between pre and interval IV thresholds at 500, 1,000, and 2,000 Hz . In the non-operated ear, we noted statistically significant changes between pre and interval V thresholds at frequencies of 4,000 and 8,000 Hz  Fig. In terms of AC thresholds in the implanted ear, the analysis shows significant differences between hearing thresholds at 250 Hz before and after implantation up to 1 year after FMT implantation and over each interval . However, in all cases, it disappeared within 3 months of surgery. One patient reported periodic vertigo. In two cases, when there was a sudden deterioration in hearing (while the VSB device was still working properly), it was necessary to perform revision surgery to reposition the FMT. After that procedure, hearing with the VSB device was restored. The incidence of adverse effects did not differ from other similar reports.AC threshold shifts for 250 Hz in the operated ear during the first year after surgery suggest that it might be due to the healing process in the middle ear after FMT implantation. After 12 months, AC threshold shifts were no longer significant.The nature of other significant AC threshold changes is difficult to interpret, and the authors believe that they might be related to the character of conductive and mixed hearing loss, which usually shows periodic fluctuations. The changes may also reflect some other unidentified phenomena occurring in the middle ear after implantation.Significant changes in BC thresholds observed within the first two intervals for 2,000 and 4,000 Hz may be the effect of healing after surgery. It suggests that clear indications of hearing preservation after direct FMT implantation against the RW without interposed fascia cannot be seen until 3 months after surgery. However, the results of BC thresholds over the 36-month follow-up confirm full hearing preservation after direct implantation of the FMT against the RW.


A case of a 31-year-old male who developed profound sensorineural hearing loss following a heroin overdose is presented. The patient subsequently had a full recovery of his hearing. Other cases of this rare phenomenon are reviewed and management options are discussed. There are only a few cases of heroin overdose associated hearing loss reported in the literature –5. More A 31-year-old male with a history of opiate abuse, including previous overdoses, presented to the emergency department of an outside hospital after being found unresponsive by his family. Heroin was found at his bedside by his family. On presentation to the emergency department, he had pinpoint pupils, was hypotensive, and was difficult to arouse. He was given Naloxone and resuscitated with intravenous fluids.As his mental status improved, he complained of new onset bilateral hearing loss, abdominal pain, and nausea. His urine toxicology was positive for opiates and negative for all other substances. His blood chemistry was consistent with rhabdomyolysis with a creatinine of 1.4, lactate of 3.2, CPK of 2,397, and potassium of 6.0. He was admitted to the critical care unit for further management. A CT Head was performed that showed infratemporal air but was otherwise normal with no evidence of infarct. At the outside hospital, he was also given one dose of IV solumedrol 120 mg. He was stabilized and transferred to our institution for further evaluation. A formal audiogram was performed, which demonstrated moderate-to-severe bilateral sensorineural hearing loss. On his audiogram, he was noted to have very poor word discrimination bilaterally . His tymOne week later, he was seen as an outpatient. He reported significant improvement in his hearing, noting greater improvement on the left than, the right. He noted some tinnitus and fullness in his right ear but denied any other otologic symptoms. His audiogram demonstrated mild, mixed, and mid frequency on the right and mild sensorineural hearing loss on the left at 3000 hz . His worSudden sensorineural hearing loss after intravenous heroin overdose has been described in five previous cases –5. In alThe mechanism of heroin overdose associated hearing loss is not well understood. Several theories have been proposed to explain this phenomenon.Like the patient in our case, many of these patients are hypotensive on presentation. Previous studies have shown a possible link between hypotension and sensorineural hearing loss , 10. HowStudies in rats and guinea pigs have found the presence of mu, kappa, and delta opioid receptors on the cochlea , 12. ActAnother possible explanation is that the heroin was mixed with an ototoxic drug. Heroin has been reported to be mixed with quinine. Quinine toxicity is a well-known cause of reversible high frequency hearing loss. There have been cases of quinine toxicity effects being seen in patients after injection of what they thought was heroin , 16.At this time, the complete explanation of opiate induced hearing loss is not known. Although steroids have been used for treatment in other cases, there is not sufficient evidence supporting their use. Fortunately in our case and in most of the other cases reported in the literature, the hearing loss was reversible and patients recovered. Future research examining the effects of opiates on the functions of the inner ear is needed to better understand the physiological process and response to these drugs.


Purpose. Subjective tinnitus has different forms and degrees of severity. Many studies in the literature have assessed psychoacoustic characteristics of tinnitus but hardly any of them had focused on the association of audiological profile with onset duration and loudness perception. The aim of this study was to evaluate existence of any association between tinnitus loudness/onset duration and audiological profile to explain differences in prognosis. Method. Study design was prospective. The sample consisted of 26 subjects having tinnitus, which was divided into tinnitus and nontinnitus ears. Audiological profile included pure-tone audiometry, speech audiometry, tympanometry, acoustic reflex test, and auditory evoked potentials (early and middle latency). Unpaired t-test was applied to compare two subgroups. Correlation and association between tinnitus onset duration/loudness perception and audiological profile were also assessed by calculating Spearman's coefficient and Fischer exact value. Results. The two subgroups had significant differences for pure-tone and speech audiometry hearing thresholds. A significant association was observed between the high frequency/extended high frequency and tinnitus loudness/onset duration. Conclusion. The changes in hearing thresholds and auditory pathway are associated with an increase in tinnitus loudness and its onset duration. This knowledge would be helpful to differentiate between severity and chronicity of the patients for planning therapeutic management and predicting prognosis. Subjective tinnitus is the perception of sounds by the patient without any physical presence of acoustic stimulus . The perSubjective ratings of tinnitus loudness, using visual analogue scales, have been found to correlate with distress , althougMany aspects of tinnitus are yet to be answered completely as what are the differences in tinnitus ear and nontinnitus ear; how the loudness perceived is relevant to treatment; whether it is associated with auditory changes; whether the onset was recent or long standing; and how these differences affect the prognosis of tinnitus. It was observed in our tinnitus therapy clinics that these differences seemed to play some role in prognosis and this information might help in planning focused and effective management of the subjects with tinnitus. Therefore the objective of the present study was to find any possible correlation of the perceived loudness and onset duration of tinnitus with audiological profile and its role in treatment. The null hypothesis was made of no correlation between the variables and had no impact on treatment plan.This was a nonrandomized study which included 30 subjects who were seeking treatment for their tinnitus problem in outdoor patient services of the institute. A prior approval of the institute ethics committee was obtained before commencement of the study. Due to the time constraints of the study, a small sample was selected and consisted of subjects of either sex from rural and urban backgrounds. There was a dropout of 4 subjects; hence analysis was done with 26 subjects. All the subjects had chief complaint of idiopathic subjective tinnitus with or without hearing loss. The inclusion criteria were healthy external or middle ear on clinical examination, consistent tinnitus of more than 10-week duration, and those who gave their written consent for the study. Patients with external or middle ear pathology, suspected Meniere's disease or otosclerosis, history of ototoxicity, sudden hearing loss, or ear trauma or having any systemic disorders were excluded.The cases were subjected to a complete clinical and audiological assessment. The audiological assessment consisted of pure-tone audiometry , high frequency audiometry (HFA), extended high frequency audiometry (EHA), speech audiometry (speech reception threshold (SRT), speech discrimination score (SDS), most comfortable level (MCL), and uncomfortable level (UCL)), Tympanometry, acoustic reflex testing (ART) , auditory brainstem responses (ABR), middle latency responses (MLR), tinnitus matching (pitch and loudness), and residual inhibition (RI). Otoacoustic emission (OAEs) and magnetic resonance imaging (MRI) were done in few selected cases. The different equipment used for the audiological investigations included Madsen orbiter 922 clinical audiometer, Siemens SD 30 tympanometer, evoked potential system developed by Intelligent Hearing System, USA, and otoacoustic system developed by Intelligence Hearing System, USA. The assessment also included subjective scaling  of annoyance and sleep disturbance due to tinnitus. These investigations were done in two visits of 50–60 minutes each prior to start of treatment. The management was planned as combination of masking therapy, environment enrichment with music, and cognitive behavior therapy.Statistical Analysis. The data was subjected to the values of mean and median for central tendency and standard deviation (SD) for variability. Unpaired t-test was applied for comparison of tinnitus and nontinnitus ears. Spearman's correlation coefficient (rho) was calculated between psychoacoustic characteristics and the audiological profile of tinnitus ears. Fischer's exact test was used as a nonparametric inferential test to verify any association between tinnitus onset duration and type of hearing loss. Similarly it was also used to verify any association of tinnitus loudness with the type of hearing loss. All significance tests were two tailed and conducted at or above the 95% significance level (P < 0.05).The 26 subjects included in the study ranged from 16 to 45 years of age with a mean 37.12 years ± SD of 8.57. Males and females were equally distributed. The psychoacoustic characteristics included tinnitus ear, duration of tinnitus since onset, pitch matching, loudness matching, and residual inhibition of tinnitus. 28% of the subjects had tinnitus in the right ear, 40% had in the left ear, and 32% had tinnitus perception binaurally. The sample was divided into two groups, tinnitus ears (35 ears) and nontinnitus ears (17 ears).The majority of tinnitus ears (72%) had onset duration longer than six months. 22.9% of the ears had duration of tinnitus perception <0.5 yrs (2.5 months–6 months), 25.7% of the ears had tinnitus perception since 0.5 to 1 year, 2.9% since 1.0 to 1.5 years, 14.3% since 1.5 to 2 years, 17.1% since 2.0 to 5.0 years, and 17.1% since >5 years. Tinnitus matching was done where 44% of the ears had pitch perception <6000 Hz and 48% had perception ≥6000 Hz. Loudness matching showed that 24% of the subjects had faint tinnitus perception ≤30 dB HL, 44% had loud (31 to 50 dB HL), 20% had too loud (51 to 70 dB HL), and 12% had extremely loud level (71 to 90 dB HL). Loudness matching was done contralaterally in hearing level (HL) units rather than in sensation level units (SL) . ResiduaThe level of annoyance was subjectively reported by the individuals on a 5-point scale, where 0 = no annoyance, 1 = little annoyance, 2 = average annoyance, 3 = high annoyance, and 4 = intolerable. All of the subjects reported level of annoyance as average or more than average, that is, ≥2. The sleep disturbance due to tinnitus was rated on a 5-point scale as 0 = never affected, 1 = rarely affected, 2 = sometimes affected, 3 = mostly affected, and 4 = always affected. Out of the total 26 subjects, 22 reported sleep disturbance as sometimes affected due to tinnitus, two of the patients reported as mostly affected (since <6 weeks), and one reported as always affected (since 2 weeks).Based on pure-tone audiometric thresholds, PTA1  was calculated for both right and left ears , and hearing status of the subjects was categorized into mild, moderate, moderate to severe, severe, and profound  . SimilarThe data shows that 60% (21 ears) of tinnitus ears had normal hearing on conventional audiometry (PTA1), 20% (7) ears had mild hearing loss, 11.4% (4) had moderate hearing loss, and 8.6% (3) of tinnitus ears had severe hearing loss. None of the tinnitus ears had profound hearing loss . The datOn high frequency audiometry (PTA2), 34.3% (12 ears) of the tinnitus ears had normal hearing, 17.1% (6 ears) had mild hearing loss, 14.3% (5) ears had moderate hearing loss, 17.1% (6) ears had moderate to severe hearing loss, 11.4% (4) ears had severe hearing loss, and 5.7% (2) of the tinnitus ears had profound hearing loss . For nonAccording to extended high frequency audiometry (PTA3), 5.7% (2 ears) of the tinnitus ears had normal hearing, 20% (7) ears had mild hearing loss, 17.1% (6) ears had moderate hearing loss, 28.6% (10) ears had moderate to severe hearing loss, 17.1% (6) ears had severe hearing loss, and 11.4% (4) of the tinnitus ears had profound hearing loss. For nontinnitus ears on PTA3, 23.5% (4) had normal hearing, 35.5% (6) ears had mild hearing loss, 17.6% (3) ears had moderate hearing loss, 17.6% (3) ears had moderate to severe hearing loss, and 5.8% (1) ear had profound hearing loss . The speech reception threshold (SRT) was normal, that is, ≤25 dBHL in 57.1% (20 ears) of the tinnitus ears, and 17.1% (6) of the tinnitus ears had SRT between 26 and 40 dBHL. It shows that the majority (74.2%) of tinnitus ears had good speech reception at general conversational levels . Speech The SRT was normal, that is, ≤25 dBHL in 82.3% (14ears) of the nontinnitus ears, and the rest 17.64% (3) of the ears had SRT between 26 and 40 dBHL . Speech The interpeak latency (IPL) of wave I-III was considered normal as 1.6–2.4 ms, shortened as <1.6 ms, and prolonged as >2.4 ms. Wave III-V IPL was normal as 1.8–2.2 ms, shortened as <1.8 ms, and prolonged as >2.2 ms. The IPL of wave I–V was normal as 3.6–4.4 ms, shortened as <3.6 ms, and prolonged as >4.4 ms. The correction (0.1 ms for every 10 dB of hearing loss above 50 dB) was applied to calculate the absolute latency of wave V when the subject had hearing loss greater than 50 dBHL at 4000 Hz .The IPL of wave I–III was observed as normal in 82.8% (29 ears) of the tinnitus ears, prolonged in 8.6% (3) ears, and no response in 8.6% (3) ears. None of the ears had shortened wave I–III interpeak latency. In non-tinnitus interpeak latency of wave I–III was normal in 94.1% of the ears (16 ears), and prolonged in 5.9% (1) of the ears. None of the ears had shortened IPL of wave I–III. Wave III–V interpeak latency was normal in 17 tinnitus ears (48.6%), shortened in 16 ears (45.7%), and prolonged in one tinnitus ear (2.8%) and there was no response in one ear (2.8%). In non-tinnitus ears wave III–V was observed as normal in 10 ears (58.8%) and shortened in seven ears (41.2%). Data shows IPL of wave I–V as normal in 30 tinnitus ears (85.7%), shortened in one ear (2.8%), prolonged in one ear (2.8%), and no response in three ears (8.6%). IPL of wave I–V in non-tinnitus was normal in 88.2% (15) of the ears, shortened in 5.9% (1), ears and prolonged in 5.9% (1) of the ears. MRI was normal in this one subject. Magnetic resonance imaging (MRI) was recommended in subjects with unexplained prolonged IPL of waves to ensure no retrocochlear pathology. Those with retrocochlear pathology were excluded from the study.μV and abnormal as <0.50 μV.Peaks observed during MLR were Na, Pa and Nb waves, out of these amplitude of waves Na and Pa were analyzed. The amplitude of waves Na and Pa was considered normal as ≥0.50 Wave Na had normal amplitude in 94.3% (33 ears) of the tinnitus ears, abnormally low in 2.8% (1) of the ears, and no response in 2.8% (1) of the tinnitus ears. Amplitude of wave Pa was normal in 32 ears (91.4%), abnormal in 2 ears (5.7%), and of no response in 1 tinnitus ear (2.8%). In non-tinnitus the amplitude of waves Na and Pa was observed to be as normal in all of the 17 ears (100%).t”-test. The comparison was made between all the measured audiological parameters, but the results of statistically significant findings are depicted in the tables. The two groups tinnitus and non-tinnitus ears were compared with unpaired “Positive correlation was observed between tinnitus onset duration and high frequency thresholds . Similarly positive correlation was also observed for extended high frequency threshold (12000 and 14000 and EHF average PTA3). Positive correlation was also observed between tinnitus duration and absolute latencies of waves III and V of ABR .Negative correlation was observed between duration and speech discrimination score (SDS) in tinnitus ears. None of the correlation coefficient values was statistically significant between tinnitus duration and interpeak latencies of ABR waves. Similarly, none of the correlation coefficient values was statistically significant between duration and amplitude of middle latency evoked response (MLR) waves Na and Pa.Significant correlation was observed between perceived tinnitus loudness and conventional audiometric thresholds and average. Similarly significant correlation was found for high frequency and extended high frequency thresholds. As shown in f = 21.149, P < 0.05) was found between duration and conventional hearing loss (PTA1). Similarly significant value  between duration and high frequency hearing loss (PTA2) was observed. Association of duration with extended high frequency hearing loss (PTA3) (f = 24.25) was nonsignificant.Fischer's exact test was used to evaluate the association between tinnitus onset duration and type of hearing loss. Statistically significant association value . Association was also statistically significant  between tinnitus loudness and high frequency hearing loss. It was nonsignificant for extended high frequency hearing loss (f = 18.507).Fischer's exact test was also used to assess association of perceived tinnitus loudness with type of hearing loss. Association was significant between loudness and conventional hearing loss (PTA1)  and individual cognitive behavior therapy (CBT) was given for two weeks. Improvement was defined objectively as change in loudness level ≥15 dBHL plus subjectively as improvement on a 5-point scale of annoyance rating and a 5-point scale of sleep disturbance. When the loudness level was faint or loud and onset duration was <0.5 years, the prognosis reported by the subjects was good immediately after completing therapy and six months later. Given similar management the subjects with loudness perception of 51 dB HL to 70 db HL (too loud) and/or onset duration 0.5–1 year reported fair improvement immediately after the therapy and some relapse by 2-3 months hence needed further therapeutic management. When onset duration of tinnitus was longer than 2 years and/or loudness was 71−90 dbHL (extremely loud) with this therapeutic plan, there was hardly any improvement and the subjects were shifted to other management strategies after 2 weeks like combination with electrical stimulation.In the study, the majority (60% of tinnitus ears and 80% of non-tinnitus ears) of the subjects had normal hearing on conventional pure-tone average (PTA1) which goes in accordance with the results published by Roberts et al. . Other sHearing thresholds were poorer in high frequency region 4000, 8000, and 12000 Hz. Roberts et al.  also repMost of the tinnitus subjects in our study had hearing loss in both ears by extended high frequency (EHF) average (94.2% of tinnitus ears and 76.5% of non-tinnitus ears). However, on comparison of tinnitus and non-tinnitus ears, significant differences were observed for extended high frequency hearing thresholds and PTA3. Barnea et al.  found thABR studies in the literature do not reveal any shortening of IPL I–III, III–V, or I–V waves. However, we found wave III–V interpeak latency shortening (<1.8 msec) in many subjects (45.7% in tinnitus ears and 41.2% in non-tinnitus ears). This indicates lesser conduction time of auditory stimulus at higher brainstem level. Moller  studied It therefore becomes clear that tinnitus perception is associated with changes in auditory pathway especially the areas responsible for high frequencies and extended high frequencies processing. And these changes are correlated with prolongation of time and increase in intensity of tinnitus perception.According to psychological models, promising treatment for tinnitus is to modify the central nervous substrate of tinnitus and consequently its percept . Tyler  reportedThis study has some limitations which could be addressed in future studies. Though a thorough case history of the subjects was taken, the possibility of pretinnitus hearing loss or audiological problems cannot be fully ruled out. Inclusion of radiological investigations particularly functional MRI would have been beneficial and would have added to the validity of the changes. The bigger sample would be safe to generalize the findings.The duration and/or loudness of tinnitus perception has strong association with changes of auditory system. There might be progressive changes on hearing and auditory pathway due to longer onset duration/tinnitus loudness or vice versa. This information should be collected during assessment to be used for planning management in a focused and effective manner. It might also be used for predicting prognosis.