Light sheet microscopy of the gerbil cochlea Hutson, Kendall A.; Pulver, Stephen H.; Ariel, Pablo ...
Journal of comparative neurology (1911),
March 2021, Volume:
529, Issue:
4
Journal Article
Peer reviewed
Open access
Light sheet fluorescence microscopy (LSFM) provides a rapid and complete three‐dimensional image of the cochlea. The method retains anatomical relationships—on a micrometer scale—between internal ...structures such as hair cells, basilar membrane (BM), and modiolus with external surface structures such as the round and oval windows. Immunolabeled hair cells were used to visualize the spiraling BM in the intact cochlea without time intensive dissections or additional histological processing; yet material prepared for LSFM could be rehydrated, the BM dissected out and reimaged at higher resolution with the confocal microscope. In immersion‐fixed material, details of the cochlear vasculature were seen throughout the cochlea. Hair cell counts (both inner and outer) as well as frequency maps of the BM were comparable to those obtained by other methods, but with the added dimension of depth. The material provided measures of angular, linear, and vector distance between characteristic frequency regions along the BM. Thus, LSFM provides a unique ability to rapidly image the entire cochlea in a manner applicable to model and interpret physiological results. Furthermore, the three‐dimensional organization of the cochlea can be studied at the organ and cellular level with LSFM, and this same material can be taken to the confocal microscope for detailed analysis at the subcellular level.
Light sheet microcopy is used to map frequency along the basilar membrane within intact cochleae, retaining spatial orientation and relationships with internal and external structural landmarks. Three‐dimensional measures of how frequency domains relate to one another gives insight into how the geometric organization of the cochlea may influence its physiology.
The need to develop new tools and increase capacity to test pharmaceuticals and other chemicals for potential adverse impacts on human health and the environment is an active area of development. ...Much of this activity was sparked by two reports from the US National Research Council (NRC) of the National Academies of Sciences, Toxicity Testing in the Twenty-first Century: A Vision and a Strategy (2007) and Science and Decisions: Advancing Risk Assessment (2009), both of which advocated for “science-informed decision-making” in the field of human health risk assessment. The response to these challenges for a “paradigm shift” toward using new approach methodologies (NAMS) for safety assessment has resulted in an explosion of initiatives by numerous organizations, but, for the most part, these have been carried out independently and are not coordinated in any meaningful way. To help remedy this situation, a framework that presents a consistent set of criteria, universal across initiatives, to evaluate a NAM's fit-for-purpose was developed by a multi-stakeholder group of industry, academic, and regulatory experts. The goal of this framework is to support greater consistency across existing and future initiatives by providing a structure to collect relevant information to build confidence that will accelerate, facilitate and encourage development of new NAMs that can ultimately be used within the appropriate regulatory contexts. In addition, this framework provides a systematic approach to evaluate the currently-available NAMs and determine their suitability for potential regulatory application. This 3-step evaluation framework along with the demonstrated application with case studies, will help build confidence in the scientific understanding of these methods and their value for chemical assessment and regulatory decision-making.
•3-step framework to focus a NAM's evaluation to criteria of higher importance.•Criteria will vary on a sliding scale in importance with changes in context-of-use and global regulatory considerations.•Case studies evaluate the NAMs for hazard screening from different approaches (e.g. in vitro, in silco, computational).
Electrocochleography (ECochG) is a potential clinically valuable technique for predicting speech perception outcomes in cochlear implant (CI) recipients, among other uses. Current analysis is limited ...by an inability to quantify hair cell and neural contributions which are mixed in the ongoing part of the response to low frequency tones. Here, we used a model based on source properties to account for recorded waveform shapes and to separate the combined signal into its components. The model for the cochlear microphonic (CM) was a sinusoid with parameters for independent saturation of the peaks and the troughs of the responses. The model for the auditory nerve neurophonic (ANN) was the convolution of a unit potential and population cycle histogram with a parameter for spread of excitation. Phases of the ANN and CM were additional parameters. The average cycle from the ongoing response was the input, and adaptive fitting identified CM and ANN parameters that best reproduced the waveform shape. Test datasets were responses recorded from the round windows of CI recipients, from the round window of gerbils before and after application of neurotoxins, and with simulated signals where each parameter could be manipulated in isolation. Waveforms recorded from 284 CI recipients had a variety of morphologies that the model fit with an average
of 0.97 ± 0.058 (standard deviation). With simulated signals, small systematic differences between outputs and inputs were seen with some variable combinations, but in general there were limited interactions among the parameters. In gerbils, the CM reported was relatively unaffected by the neurotoxins. In contrast, the ANN was strongly reduced and the reduction was limited to frequencies of 1,000 Hz and lower, consistent with the range of strong neural phase-locking. Across human CI subjects, the ANN contribution was variable, ranging from nearly none to larger than the CM. Development of this model could provide a means to isolate hair cell and neural activity that are mixed in the ongoing response to low-frequency tones. This tool can help characterize the residual physiology across CI subjects, and can be useful in other clinical settings where a description of the cochlear physiology is desirable.
The cochlear summating potential (SP) to a tone is a baseline shift that persists for the duration of the burst. It is often considered the most enigmatic of cochlear potentials because its magnitude ...and polarity vary across frequency and level and its origins are uncertain. In this study, we used pharmacology to isolate sources of the SP originating from the gerbil cochlea. Animals either had the full complement of outer and inner hair cells (OHCs and IHCs) and an intact auditory nerve or had systemic treatment with furosemide and kanamycin (FK) to remove the outer hair cells. Responses to tone bursts were recorded from the round window before and after the neurotoxin kainic acid (KA) was applied. IHC responses were then isolated from the post-KA responses in FK animals, neural responses were isolated from the subtraction of post-KA from pre-KA responses in NH animals, and OHC responses were isolated by subtraction of post-KA responses in FK animals from post-KA responses in normal hearing (NH) animals. All three sources contributed to the SP; OHCs with a negative polarity and IHCs and the auditory nerve with positive polarity. Thus the recorded SP in NH animals is a sum of contributions from different sources, contributing to the variety of magnitudes and polarities seen across frequency and intensity. When this information was applied to observations of the SP recorded from the round window in human cochlear implant subjects, a strong neural contribution to the SP was confirmed in humans as well as gerbils.
Of the various potentials produced by the cochlea, the summating potential (SP) is typically described as the most enigmatic. Using combinations of ototoxins and neurotoxins, we show contributions to the SP from the auditory nerve and from inner and outer hair cells, which differ in polarity and vary in size across frequency and level. This complexity of sources helps to explain the enigmatic nature of the SP.
OBJECTIVES:Electrocochleography (ECochG) obtained through a cochlear implant (CI) is increasingly being tested as an intraoperative monitor during implantation with the goal of reducing surgical ...trauma. Reducing trauma should aid in preserving residual hearing and improve speech perception overall. The purpose of this study was to characterize intracochlear ECochG responses throughout insertion in a range of array types and, when applicable, relate these measures to hearing preservation. The ECochG signal in cochlear implant subjects is complex, consisting of hair cell and neural generators with differing distributions depending on the etiology and history of hearing loss. Consequently, a focus was to observe and characterize response changes as an electrode advances.
DESIGN:In 36 human subjects, responses to 90 dB nHL tone bursts were recorded both at the round window (RW) and then through the apical contact of the CI as the array advanced into the cochlea. The specific setup used a sterile clip in the surgical field, attached to the ground of the implant with a software-controlled short to the apical contact. The end of the clip was then connected to standard audiometric recording equipment. The stimuli were 500 Hz tone bursts at 90 dB nHL. Audiometry for cases with intended hearing preservation (12/36 subjects) was correlated with intraoperative recordings.
RESULTS:Successful intracochlear recordings were obtained in 28 subjects. For the eight unsuccessful cases, the clip introduced excessive line noise, which saturated the amplifier. Among the successful subjects, the initial intracochlear response was a median 5.8 dB larger than the response at the RW. Throughout insertion, modiolar arrays showed median response drops after stylet removal while in lateral wall arrays the maximal median response magnitude was typically at the deepest insertion depth. Four main patterns of response magnitude were seenincreases > 5 dB (12/28), steady responses within 5 dB (4/28), drops > 5 dB (from the initial response) at shallow insertion depths (< 15 mm deep, 7/28), or drops > 5 dB occurring at deeper depths (5/28). Hearing preservation, defined as < 80 dB threshold at 250 Hz, was successful in 9/12 subjects. In these subjects, an intracochlear loss of response magnitude afforded a prediction model with poor sensitivity and specificity, which improved when phase, latency, and proportion of neural components was considered. The change in hearing thresholds across cases was significantly correlated with various measures of the absolute magnitudes of response, including RW response, starting response, maximal response, and final responses (p’s < 0.05, minimum of 0.0001 for the maximal response, r’s > 0.57, maximum of 0.80 for the maximal response).
CONCLUSIONS:Monitoring the cochlea with intracochlear ECochG during cochlear implantation is feasible, and patterns of response vary by device type. Changes in magnitude alone did not account for hearing preservation rates, but considerations of phase, latency, and neural contribution can help to interpret the changes seen and improve sensitivity and specificity. The correlation between the absolute magnitude obtained either before or during insertion of the ECochG and the hearing threshold changes suggest that cochlear health, which varies by subject, plays an important role.
Almost all patients who receive cochlear implants have some acoustic hearing prior to surgery. Electrocochleography (ECoG), or electrophysiological measures of cochlear response to sound, can ...identify remaining auditory nerve activity that is the basis for this residual hearing and can record potentials from hair cells that are no longer functionally connected to nerve fibers. The ECoG signal is therefore complex, being composed of both hair cell and neural signals. To identify signatures of different sources in the recorded potentials, we collected ECoG data across frequency and intensity from the round window of gerbils before and after treatment with kainic acid, a neurotoxin. Distortions in the recorded waveforms were produced by different sources over different ranges of frequency and intensity. In response to tones at low frequencies and low-to-moderate intensities, the major source of distortion was from neural phase-locking that was sensitive to kainic acid. At high intensities at all frequencies, the distortion was not sensitive to kainic acid and was consistent with asymmetric saturation of the hair cell transducer current. In addition to loss of phase-locking, changes in the envelope after kainic acid treatment indicate that sustained neural firing combines with receptor potentials from hair cells to produce the envelope of the response to tones. These results provide baseline data to interpret comparable recordings from human cochlear implant recipients.
Objective/Hypothesis
Previous reports have documented the feasibility of utilizing electrocochleographic (ECoG) responses to acoustic signals to assess trauma caused during cochlear implantation. The ...hypothesis is that intraoperative round window ECoG before and after electrode insertion will help predict postoperative hearing preservation outcomes in cochlear implant recipients.
Study Design
Prospective cohort study.
Methods
Intraoperative round window ECoG responses were collected from 31 cochlear implant recipients (14 children and 17 adults) immediately prior to and just after electrode insertion. Hearing preservation was determined by postoperative changes in behavioral thresholds.
Results
On average, the postinsertion response was smaller than the preinsertion response by an average of 4 dB across frequencies. However, in some cases (12 of 31) the response increased after insertion. The subsequent hearing loss was greater than the acute loss in the ECoG, averaging 22 dB across the same frequency range (250–1,000 Hz). There was no correlation between the change in the ECoG response and the corresponding change in audiometric threshold.
Conclusions
Intraoperative ECoG is a sensitive method for detecting electrophysiologic changes during implantation but had limited prognostic value regarding hearing preservation in the current conventional cochlear implant patient population where hearing preservation was not intended.
Level of Evidence
2b Laryngoscope, 126:1193–1200, 2016
Characterize the contribution of the auditory nerve neurophonic (ANN) to electrocochleography (ECochG) of pediatric cochlear implant (CI) recipients with and without auditory nerve spectrum disorder ...(ANSD).
ECochG is an emerging technique for predicting outcomes in CI recipients. Its utility may be increased by separating the cochlear microphonic (CM), produced by hair cells, from the ANN, the evoked potential correlate of neural phase-locking, which are mixed in the ongoing portion of the response to low frequency tone bursts.
Responses to tone bursts of different frequency and intensities were recorded from the round window of pediatric CI recipients. Separation of the CM and ANN was performed using a model of the underlying processes that lead to the shapes of the observed waveforms.
Preoperative mean pure tone amplitudes of the included ANSD (n = 36) and non-ANSD subjects (n = 123), were similar (89.5 and 93.5, p = 0.1). Total of 1,024 ECochG responses to frequency and intensity series were recorded. The mean correlation ( r ) between the input and the modeled signals was 0.973 ± 0.056 (standard deviation). The ANN magnitudes were higher in the ANSD group (ANOVAs, F = 26.5 for frequency and 21.9 for intensity, df's = 1, p 's < 0.001). However, its relative contribution to the overall signal was lower (ANOVAs, F = 25.8 and 12.1, df = 1, p 's < 0.001).
ANN was detected in low frequency ECochG responses but not high frequency responses in both ANSD and non-ANSD subjects. ANSD subjects, evidence of neural contribution in responses to low frequency stimuli was highly variable and often comparable to signals recorded in non-ANSD subjects. The computational model revealed that on average the ANN comprised a lower proportion of the overall signal than in non-ANSD subjects.
To investigate the electrophysiology of the cochlear summating potential (SP) in patients with Meniere's disease (MD). Although long considered a purely hair cell potential, recent studies show a ...neural contribution to the SP. Patients with MD have an enhanced SP compared to those without the disease. Consequently, this study was to determine if the enhancement of the SP was in whole or part due to neural dysfunction.
Study participants included 41 adults with MD and 53 subjects with auditory neuropathy spectrum disorder (ANSD), undergoing surgery where the round window was accessible. ANSD is a condition with known neural dysfunction, and thus represents a control group for the study. The ANSD subjects and 17 of the MD subjects were undergoing cochlear implantation (CI) surgery; the remaining MD subjects were undergoing either endolymphatic sac decompression or labyrinthectomy to alleviate the symptoms of MD. Electrocochleography was recorded from the round window using high intensity (90 dB nHL) tone bursts. The SP and compound action potential (CAP) were measured to high frequencies (> = 2 kHz) and the SP, cochlear microphonic (CM) and auditory nerve neurophonic (ANN) to low frequencies. Linear mixed models were used to assess differences between MD and ANSD subjects.
Across frequencies, the MD subjects had smaller alternating current (AC) response than the ANSD subjects (
= 31.6
,
,
< 0.001), but the SP magnitudes were larger (
= 94.3
,
,
< 0.001). For frequencies less than 4 kHz the SP magnitude in the MD group was significantly correlated with the magnitude of the CM (
's < 0.001) but not in the ANSD group (
's > 0.05). Finally, the relative proportions of both ANN and CAP were greater in MD compared to ANSD subjects. The shapes of the waveforms in the MD subjects showed the presence of multiple components contributing to the SP, including outer and inner hair cells and neural activity.
The results support the view that the increased negative polarity SP in MD subjects is due to a change in the operating point of hair cells rather than a loss of neural contribution. The steady-state SP to tones in human subjects is a mixture of different sources with different polarities.
OBJECTIVES:Variability in speech perception outcomes with cochlear implants remains largely unexplained. Recently, electrocochleography, or measurements of cochlear potentials in response to sound, ...has been used to assess residual cochlear function at the time of implantation. Our objective was to characterize the potentials recorded preimplantation in subjects of all ages, and evaluate the relationship between the responses, including a subjective estimate of neural activity, and speech perception outcomes.
DESIGN:Electrocochleography was recorded in a prospective cohort of 284 candidates for cochlear implant at University of North Carolina (10 months to 88 years of ages). Measurement of residual cochlear function called the “total response” (TR), which is the sum of magnitudes of spectral components in response to tones of different stimulus frequencies, was obtained for each subject. The TR was then related to results on age-appropriate monosyllabic word score tests presented in quiet. In addition to the TR, the electrocochleography results were also assessed for neural activity in the forms of the compound action potential and auditory nerve neurophonic.
RESULTS:The TR magnitude ranged from a barely detectable response of about 0.02 µV to more than 100 µV. In adults (18 to 79 years old), the TR accounted for 46% of variability in speech perception outcome by linear regression (r = 0.46; p < 0.001). In children between 6 and 17 years old, the variability accounted for was 36% (p < 0.001). In younger children, the TR accounted for less of the variability, 15% (p = 0.012). Subjects over 80 years old tended to perform worse for a given TR than younger adults at the 6-month testing interval. The subjectively assessed neural activity did not increase the information compared with the TR alone, which is primarily composed of the cochlear microphonic produced by hair cells.
CONCLUSIONS:The status of the auditory periphery, particularly of hair cells rather than neural activity, accounts for a large fraction of variability in speech perception outcomes in adults and older children. In younger children, the relationship is weaker, and the elderly differ from other adults. This simple measurement can be applied with high throughput so that peripheral status can be assessed to help manage patient expectations, create individually-tailored treatment plans, and identify subjects performing below expectations based on residual cochlear function.
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