Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
The authors test the "OHC-fluid-pump" hypothesis by assaying the rates of kainic acid dispersal both in quiet and in cochleae stimulated by sounds of different levels and spectral content. The main result is that sound (and thus, presumably, OHC contractions and expansions) results in faster transport along the duct. OHC involvement is corroborated using salicylate, which yielded results similar to silence. Especially interesting is the fact that some stimuli (e.g. tones) seem to provide better/faster pumping than others (e.g. noise), ostensibly due to the phase profile of the resulting cochlear traveling-wave response.
Strengths:
The experiments appear well controlled and the results are novel and interesting. Some elegant cochlear modeling that includes coupling between the organ of Corti and the surrounding fluid as well as advective flow supports the proposed mechanism.
Weaknesses:
It's not clear whether the effect size (e.g., the speed of sound-induced pumping relative to silence) is large enough to have important practical applications (e.g., for drug delivery). The authors should comment on the practical requirements and limitations.
With our current data, what we can conclude is that modest sound levels (e.g., 75 dB SPL noise or an 80 dB SPL tone) facilitates cochlear drug delivery. We added a paragraph to the Discussion stating some future considerations for application to drug delivery in the human cochlea.
Although helpful so far as it goes, the modeling could be taken much further to help understand some of the more interesting aspects of the data and to obtain testable predictions. In particular, the authors should systematically explore the level effects they find experimentally and determine whether the model can replicate the finding that different sounds produce different results (e.g. noise vs tone).
The model should also be used to relate the model's flow rates more quantitatively to the properties of the traveling wave (e.g., its phase profile).
The present study is focused on explaining the principle of mass transport in the cochlea. The quantification of the relationship between flow rate and traveling wave is an important open question and will be the topic of future studies. Our previous modeling study (Shokrian et al. 2020) showed a clear relation between the traveling wave characteristics (e.g., amplitude and phase velocity) and the mass transport in the Corti fluid. As the reviewer correctly pointed out, the current paper is focused on designing controlled experiments to provide proof of concept along with computational simulations to support our major claim (that outer hair cells stir cochlear fluid).
Finally, the model should be used to investigate differences between active and passive OHCs (e.g., simulating the salicylate experiment by disabling the model's OHCs).
What the reviewer asks for has been demonstrated in previous theoretical studies (Lighthill, 1992; Edom, Obrist, Kleiser, 2014; Sumner, Reichenbach, 2021). In some of the previous studies, it was called the steady streaming. These studies are excellent examples because they simulated the sensitive cochlea (similar level of basilar membrane vibrations) but did not incorporate the Corti fluid peristalsis. Even without the peristaltic motion of the Corti tube, the basilar membrane-scala fluid interaction generated steady streaming (creepy fluid flow). However, the streaming velocity of cochlear models without active peristalsis along the Corti tube is about three orders of magnitude smaller than the active cochlea at a comparable level of basilar membrane vibrations. For example, the peak streaming speed was < 0.1 um/s at 80 dB SPL, and it took > 4 hours for particles to travel 1 mm. This speed is much slower than the particle transport speed due to pure diffusion (Sumner, Reichenbach, 2021).
The manuscript would be stronger if the authors discussed ways to test their hypothesis that OHC motility serves a protective effect by pumping fluid. For example, do animals held in quiet after noise exposure (TTS) take longer to recover?
We agree with the reviewer. The following statements were added to the Discussion section. “Our results have implications for cochlear fluid homeostasis. For example, future studies can test the hypothesis that an acoustically rich environment would be beneficial in maintaining healthy hearing as well as in recovering from transient hearing loss.”
Reviewer #2 (Public review):
Summary:
Recent cochlear micromechanical measurements in living animals demonstrated outer hair celldriven broadband vibration of the reticular lamina that contradicts frequency-selective cochlear amplification. The authors hypothesized that motile outer hair cells can drive cochlear fluid circulation. This hypothesis was tested by observing the effects of acoustic stimuli and salicylate, an outer hair cell motility blocker, on kainic acid-induced changes in the cochlear nucleus activities. It was found that acoustic stimuli can reduce the latency of the kainic acid effect, and a low-frequency tone is more effective than broadband noise. Salicylate reduced the effect of acoustic stimuli on kainic acid-induced changes. The authors also developed a computational model to provide the physical basis for interpreting experimental results. It was concluded that experimental data and simulations coherently indicate that broadband outer hair cell action is for cochlear fluid circulation.
Strengths:
The major strengths of this study include its high significance and the combination of electrophysiological recording of the cochlear nucleus responses with computational modeling. Cochlear outer hair cells have been believed to be responsible for the exceptional sensitivity, sharp tuning, and huge dynamic range of mammalian hearing. Recent observation of the broadband reticular lamina vibration contradicts frequency-specific cochlear amplification. Moreover, there is no effective noninvasive approach to deliver the drugs or genes to the cochlea for treating sensorineural hearing loss, one of the most common auditory disorders. These important questions were addressed in this study by observing outer hair cells' roles in the cochlear transport of kainic acid. The well-established electrophysiological method for recording cochlear nucleus responses produced valuable new data, and the purposely developed computational model significantly enhanced the interpretation of the data.
The authors successfully tested their hypothesis, and both the experimental and modeling results support the conclusion that active outer hair cells can drive cochlear fluid circulation in the living cochlea.
Findings from this study will help auditory scientists understand how the outer hair cells contribute to cochlear amplification and normal hearing.
We thank the reviewer for acknowledging our effort.
Weaknesses:
While the statement "The present study provides new insights into the nonselective outer hair cell action (in the second paragraph of Discussion)" is well supported by the results, the authors should consider providing a prediction or speculation of how this hair cell action enhances cochlear sensitivity. Such discussion would help the readers better understand the significance of the current work.
We added a potential implication to the Discussion, that an acoustically rich environment could be beneficial in maintaining healthy hearing as well as recovering from damaged hearing.
Reviewer #3 (Public review):
Summary:
This study reveals that sound exposure enhances drug delivery to the cochlea through the nonselective action of outer hair cells. The efficiency of sound-facilitated drug delivery is reduced when outer hair cell motility is inhibited. Additionally, low-frequency tones were found to be more effective than broadband noise for targeting substances to the cochlear apex. Computational model simulations support these findings.
Strengths:
The study provides compelling evidence that the broad action of outer hair cells is crucial for cochlear fluid circulation, offering a novel perspective on their function beyond frequency-selective amplification. Furthermore, these results could offer potential strategies for targeting and optimizing drug delivery throughout the cochlear spiral.
Weaknesses:
The primary weakness of this paper lies in the surgical procedure used for drug administration through the round window. Opening the cochlea can alter intracochlear pressure and disrupt the traveling wave from sound, a key factor influencing outer hair cell activity. However, the authors do not provide sufficient details on how they managed this issue during surgery. Additionally, the introduction section needs further development to better explain the background and emphasize the significance of the work.
Although we wrote that the inner ear left intact, it might have not been sufficiently clear. Our surgical approach leaves the inner ear intact, including the round-window membrane. The round window in gerbil is concave like a bowl. We applied 4 µL of kainic acid solution in the round-window niche, without perforating the round-window membrane.
Recommendations For The Authors:
Reviewer #1 (Recommendations for the authors):
The authors' choice to frame their findings by hinting that they have discovered the "real" reason for the evolution of broadband OHC electromotility (e.g., the first and last sentences of the abstract and parts of the Discussion), although clearly intended to boost the perceived significance of the work, does them no favors and will probably lead to distracting criticisms they could easily have avoided. The manuscript would be significantly improved by removing or downplaying these rather speculative and unsupported claims; the work stands on its own without them.
We agree that the first line of the Abstract might distract the readers. Meanwhile, in the Discussion, we believe the readers will appreciate our speculation of how this study is relevant to recent debates on hearing mechanics. Following the reviewer’s advice, we have revised the Abstract.
Reviewer #3 (Recommendations for the authors):
Please review the detailed comments below. I hope they contribute to enhancing the paper:
We thank the reviewer for this detailed advice. All of these comments make good sense and were very helpful in improving this paper or in planning future studies.
Many of the comments were relevant to the computer model, and they have one common basis, which we have not yet achieved. I.e., simulating the level-dependence.
I. Introduction
(1) Please clarify and improve this sentence. Effective and safe strategies for delivering treatments to the inner ear have been reported: 'Consequently, intervening in hearing health by delivering substances to the inner-ear fluid is challenging'.
The preceding statement is regarding the blood-labyrinthine barrier (BLB), comparable to the bloodbrain barrier (BBB). We revised the statement: “Consequently, intervening in hearing health by delivering substances to the inner-ear fluid through systemic circulation is challenging.”
(2) Please expand on how the secretion and absorption of ions and molecules maintain the unique ionic compositions of the two intracochlear fluids. Include details on the role of the stria vascularis and the specific functions of the three types of strial cells in this process.
In response to this request, we added a paragraph discussing cochlear fluid homeostasis. Our study is different from existing homeostasis studies in three regards. First, the site: Existing studies are centered on the stria vascularis, while this study concerns the Corti fluid. Second, the mechanism: Existing studies are regarding metabolic transport, while our scope is the transport due to fluid flow. Third, the range: Existing studies considered local electrochemical equilibrium within a radial section, while this study concerns global (longitudinal) mass transport. To address this comment, the following was added to the Discussion.
“Our study complements existing studies regarding cochlear fluid homeostasis and differs from previous studies in several ways. The intrastrial fluids (extracellular fluids in the stria vascularis) have been more thoroughly investigated because the three layers in the stria vascularis (marginal, intermediate, and basal cells) maintain the endocochlear potential (Wangemann 2006).
Equilibrium in the Corti fluid has been sparsely investigated because its electrochemical gradient is modest compared to that of the intrastrial fluids (Johnstone, Patuzzi et al. 1989; Zidanic and Brownell 1990). Local electrochemical balance in the cochlear fluids has been considered within a radial section (Quraishi and Raphael 2008; Patuzzi 2011; Nin, Hibino et al. 2012). Our study is focused on the longitudinal (global) equilibrium along the cochlear coil and did not consider the equilibrium across the stria vascularis cell layers. To examine whether the longitudinal fluid flow driven by outer hair cells is strong enough to affect cochlear fluid homeostasis, future studies should measure the K+ equilibrium and recycling along the length of the Corti fluid under sound and silence conditions.“
(3) Please provide a more detailed explanation and definition of a longitudinal electrochemical gradient, including how it functions and its relevance in physiological processes.
The most researched electrochemical gradient of the cochlea must be the endocochlear potential that varies along the cochlear length. The endocochlear potential at any location is determined by the equilibrium between the source and the sink. In the view of the Corti fluid, the source is the potassium current out of the hair cells and the sink is the resorption of potassium by supporting cells. The effect of a longitudinal electrochemical gradient on hearing physiology is beyond the scope of this study. To do so would require incorporating detailed K+ equilibrium dynamics. This certainly is one of our future directions.
(4) Please include the necessary references to support these three sentences: "Diffusion is an effective mechanism for a substance to travel along submicrometer distances. For instance, it takes microseconds for neurotransmitters to diffuse across a 20-nm synaptic gap. In contrast, diffusion is inefficient for travel on the centimeter scale. It takes days for a drug applied at the round window to travel 30 mm to the apical end of the human cochlea. In practice, the substance would not reach the apex because it would be resorbed before traveling the distance".
A reference was added (Berg, 1993). Our description of diffusion is based on the fundamental physics of Fick’s laws.
(5) In paragraph 3, the author only discussed a portion of the previous approaches. There are numerous methods for inner ear delivery, including external, middle ear, and direct inner ear delivery via the round window or semicircular canal. Each method has its pros and cons, which the authors should carefully address. For example, the semicircular canal approach doesn't require two perforations in the inner ear and distributes the injection evenly throughout the cochlea.
A recent review paper regarding inner ear drug delivery was added as a reference (Szeto, Chiang et al. 2020). Drug delivery is a means to demonstrate the OHC’s role in longitudinal mass transport. We are concerned that comparing different drug delivery modalities in detail would distract the readers from the main point of this study. We mentioned ‘one remedy’ with two perforations, for which abundant case studies are found in the literature. Discussing existing approaches exhaustively can be better done by review papers.
(6) The following sentence is inaccurate and should be carefully rephrased. Previous reports chose higher volumes than the actual fluid volume to maximize the drug (or gene) effect, but this was not a requirement of the delivery methods: 'Such an invasive approach requires the injection of a substantial fluid volume, larger than the entire perilymph in the inner ear'.
We revised the statement to relax the wording ‘require’: ‘Such an invasive approach is often associated with the injection of a substantial fluid volume, larger than the entire perilymph in the inner ear (Szeto, Chiang et al. 2020)'. This statement might be acceptable because we found few invasive delivery papers that used < 1 µL. Moreover, the physics basis of the injection method is to replace the fluid in a labyrinth compartment with a new fluid (a good example where this fluid physics was tested with quantitative data is the Lichtenhan et al. 2016 paper).
(7) Please provide the necessary references. Also, clarify what is meant by 'actuator cells'. Are you referring to hair cells?: 'The tube-shaped organ of Corti (OoC) is lined with actuator cells and the cells are activated systematically with a large phase velocity (> a few m/s) toward the apex'.
Yes, we meant OHCs as the actuator cells. This point has been clarified. A reference for the phase velocity has been added (Olson, Duifhuis, Steele, 2012).
II. Results
(1) Is there a specific reason you use 60 or 75 dB SPL for broadband sounds, but opt for louder sounds (80 dB SPL) for pure tones?
It is not straightforward to compare the SPL between broadband noise and a pure tone, and we did not attempt to ‘equate’ them in any way.
(2) Please provide specific details about the sound generation protocol, including the duration, start time, end time, and any other relevant parameters. Here is an example of a vague sentence. Do you play the sounds continuously during these time periods, or only at specific intervals?: 'In two example cases, the effect time at low-CF locations (CFs near 2 kHz) was 15 minutes for the case of the 0.5 kHz tone (Fig. 3A)'
It is described in the Measurement protocol part of the Methods section (see the red text below). In the exampled case and all other cases, the sounds were played continually (not continuously).
For the “Sound” protocol, 1.1-s noise pips (60 or 75 dB SPL, 0.1-12 kHz bandwidth, 0.8-s duration including 0.15-s onset/offset ramps) were presented continually. After 48 noise pips, one 1.1-s silent pause and three CF tone pips followed (a total of 51 pips and a pause make a 57.2-s sequence). The CF tone pips were presented at the level of 35 dB SPL to monitor neural responses. The silence pause was to monitor spontaneous neural responses. The sequence was repeated until neural signals at the lowest CF site were completely abolished. The neural responses presented in this study are the ‘driven responses’ obtained by subtracting the spontaneous responses from the responses to the 35 dB CF tones. For the “Silence” or “Pure-tone” protocol, the noise pips of the Sound protocol were replaced with either silence pauses or a pure tone at 80 dB SPL.
(3) Providing a schematic timeline of your experiments indicating sound generation, kainic acid (and salicylate) application, as well as DPOAE and AVCN recordings would greatly help in understanding and following your results.
We have revised Figure 2.
(4) How did you control the opening(s) for the injection? The openings could alter intracochlear pressure and affect the traveling wave from the sound, which is the major factor influencing outer hair cell activity.
We did not open the inner ear. The round window remained intact. Opening the bulla does not affect the intracochlear pressure. We have clarified this issue, beginning with the first sentence of the Abstract. Thanks for raising this important question.
(5) Is there any reason why the author generated only low and mid-frequencies? If so, please address what the limitations were in testing high frequency.
There are no limitations to testing high frequencies. High frequencies would not affect drug delivery to the apex of the cochlea because the traveling waves stop right after the CF location. We are interested in delivering drugs deeper into the apex. Our presented results support this reasoning: mid-frequency stimulation was less effective for delivery to the low CF location.
(6) I suggest combining Figures 3E and 3F to facilitate a direct comparison between the Silence and Noise conditions, as the MF and LF plots are overlapping in these panels.
We considered this change but realized that it might introduce confusion and difficulty in parsing the results. Moreover, the two panels have their respective messages.
(7) In Figure 3E, why does the LF tone affect both Low and Mid CFs, while the MF tone only affects Mid CF?
The cochlear traveling wave stops right after the CF location. Peristaltic action takes place in the broad tail region of the traveling waves (see Fig. 5C).
III. Materials and Methods
(1) Please provide details about your injection protocol. Did you create additional perforations? How did you target the round window? What was the injection rate? How did you seal the round window, and so on?
The inner ear including the round window was left intact. Only the bulla was open.
(2) Please include details about your surgical procedure for the AVCN recording, including probe insertion.
AVCN recording is a well-established technique. Instead of reintroducing the method, we added a classical reference with friendlier description (Frisina, Chamberlain, et al., 1982).
IV. Minor points
(1) Please include the full terms for the abbreviations 'CF', 'DPOAEs', 'PT', 'IP', and 'RW' for readers who are not in the hearing research field.
We have checked that these abbreviations were defined.
(2) Are 'GXXX's in figures animal identifiers? Please clarify what they represent.
Yes, they are animal identifiers. We have clarified this point in Fig. 1 caption.
