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Kresge Hearing Research Institute

Department of Otolaryngology

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Cochlear Signaling and Tissue Engineering Menu

Cochlear Signaling and Tissue Engineering Laboratory

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Molecular Mechanisms and Protection in NIHL

The studies described in this section reflect work performed not only in my laboratory but also substantial efforts by Drs. Richard Altschuler, Colleen Le Prell, and Jochen Schacht, and their students. Important contributions to this work have also been made by recent post-doctoral research fellows, including Shujiro Minami, Yoshi Ohinata, Daisuke Yamashita, and Tatsuya Yamasoba.

Noise-induced hearing loss (NIHL) continues to be the primary cause of acquired hearing loss in the industrialized world. The purpose of this program of research has been to enhance our understanding of the mechanisms involved during the destruction of the sensory cells of the inner ear NIHL and to develop preventative interventions to alleviate hearing impairment as a result of noise.

NIHL is caused by a combination of three factors. The first is a direct mechanical trauma to the delicate cells and tissues of the inner ear as a result of intense vibration of these structures by loud noise. The membranes of the sensory (hair) cells and the "hairs" (stereocilia) on the hair cells are literally torn apart. This occurs at the highest levels of intense noise exposure, and can occur with very brief exposures to intense sound, e.g., an exploding firecracker. At lower exposure levels, hearing loss is also caused by an "overdriving" of the sensory cells of the hearing part of the inner ear, the cochlea. That is, due to the intense metabolic activity induced by a relatively high level of noise, over a sufficient period of time, the hair cells will undergo "oxidative stress," which can result in cell death. A third factor contributing to NIHL is vasoconstriction-induced reduction in cochlear blood flow (CBF). Research in our laboratory focuses on the latter two mechanisms: oxidative stress and reduced CBF.

Under metabolically induced oxidative stress, cells form highly reactive molecules (i.e., free radicals), which rapidly create chemical reactions with other molecules that determine the structure and function of the cell. Thus, they react with the molecules that make up the cell wall (phospholipid), changing their structure and compromising the integrity of the cell wall by literally making holes in it, and allowing the ionized form of calcium (Ca2+) to enter. Intracellular Ca2+ is critical for cell function; however, for normal cell function, it is critical to maintain Ca2+ within a narrow range of concentration. Free radicals may also react directly with DNA to produce genetic mutations. Finally, free radicals may interact with "transcription factors," which control gene expression, causing, for example, an abnormal upregulation of those genes that induce cell death.

We have shown that free radicals form in the inner ear (lateral wall, organ of Corti, and auditory nerve) during noise exposure. By measuring lipid peroxidation (free radical induced changes in the membranes of cells), we obtained a direct measure of the influence of free radicals in the inner ear following noise. Following a five-hour intense noise exposure, lipid peroxidation showed a 30-fold increase. When we examined the tissues with the greatest noise-induced lipid peroxidation with selected staining (immunohistology), we found immunostaining in the same tissues that show the greatest noise-induced increase in glutathione (an important free radical scavenger). We interpret glutathione up-regulation as an endogenous protective mechanism to "scavenge" the increase in free radicals induced by the intense noise exposure. Preventing the formation of glutathione via administration of the drug L-buthionine-[S,R]-sulfoximine (BSO) produced a major increase in NIHL and sensory cell damage in comparison to subjects that had a normal functioning scavenger system. Administration of a form of glutathione that may enter cells reduces, by approximately half, the effects of an intense noise exposure. These data suggest that administration of an agent that increases the function of normal scavenger systems or adds scavengers to the inner ear can reduce NIHL.

Recent research has shown the key role of free radical formation and reduced inner ear blood flow in NIHL. The extent, distribution, and timing of free radical formation have been defined, including a clinically significant late formation of free radicals in the 7 to 10 days following noise exposure. In addition, we have shown that by administering a combination of antioxidant agents (salicylate and trolox) prior to or up to 3 days following an exposure to intense sound, we significantly reduce threshold deficits, hair cell damage, and ROS and RNS formation. Threshold protection is illustrated in the graph below:

Threshold protection graph

Reprinted from Yamashita, et al., 2005, Neuroscience, 134, 633-642. Treatment with salicylate and Trolox initiated prior to sound trauma protects against hearing loss (measured as ABR threshold). Protective effects diminish with delay in treatment, although some protection is observed even with a 5-day delay in the onset of treatment.

Other experiments have revealed that administering a growth factor [glial derived neurotrophic factor (GDNF) or neurotrophin-3 (NT-3)], which helps to maintain intracellular Ca2+ within a normal physiologic range, can also reduce NIHL. In addition, we have found that with a combined application of GDNF plus a radical scavenger, we can observe an additive protective effect of the two agents.

Our laboratory has a long-standing interest in cochlear perfusion, having examined CBF during noise, noise effects on the cochlear vasculature and CBF and recovery, and the evaluation of agents that modify the effects of noise on the inner ear vasculature and CBF. The mechanism underlying noise-induced reduction in inner ear blood flow has finally been identified with the development of laser Doppler flowmetry as a tool for dynamic measurement of CBF. We have now shown that the long-observed anomalous noise-induced reduction in CBF is due to the formation of the bioactive product 8-iso-PGF2. Isoprostanes are known to be markers of oxidant stress and tissue damage, and some are potent vasoconstrictors. Using laser Doppler flow assessment, we demonstrated that infusion of the isoprostane 8-iso-PGF2 generates a rapid and concentration-dependent blood pressure and CBF response, followed by a recovery toward baseline over a few minutes. Other studies further demonstrate the specificity of this effect through the use of a localized vascular injection of isoprostanes at the level of the anterior inferior cerebellar artery (AICA).

Given the clear benefits of both antioxidant agents and those that preserve the blood supply to the cochlea during and after a noise insult, we recently evaluated the possibility of synergistic interactions between free radical scavengers (beta-carotene, and vitamins C and E) and one of the agents that prevents constriction of the cochlear blood supply, magnesium. Prevention of noise-induced hearing loss was significantly reduced using this dietary micronutrient treatment as illustrated in the graph below:



Reprinted from Le Prell et al. 2007, Free Rad. Biol. Med.,42, 1454-1463. A. Noise-induced hearing loss in guinea pigs, estimated using auditory brainstem response thresholds prior to and 10 days post noise (octave-band noise centered at 4 kHz, 120-dB SPL, 5 hours), was reduced by treatment with a combination of vitamins A, C, E and magnesium (ACEMg), but not by treatment with the antioxidants (ACE) or magnesium (Mg) (*) Asterisks indicate statistically reliable differences (p�s < 0.001) between ACEMg and all other groups. B. Outer hair cell loss in the 10-15 and 15-20 mm from the apex segments of the cochlea was reliably reduced when group comparisons were limited to the two groups most crucial to testing our hypothesis: saline and combination treatment with antioxidants and magnesium.

Our new mechanistic insights and findings of safe and effective interventions that attenuate NIHL provide a rich and growing scientific rationale to justify human trials to eliminate this most important cause of acquired hearing loss. We are now actively planning clinical trials with our US and international colleagues to test this combination of antioxidants and other dietary supplements in human models of NIHL, including, for example, recreational, occupational, and military noise exposures.

See the following press releases for additional NIHL information:

Auditory Nerve Survival and Regrowth

The studies described in this section reflect not only work performed in my laboratory but also substantial efforts by Drs. Richard Altschuler and Yehoash Raphael, and their students. These Directors of the Anatomy Laboratory and Otopathology (respectively) have provided invaluable help in all of these studies, in particular those involving the regrowth of peripheral processes of the auditory nerve (Altschuler) and studies of the effectiveness of adenoviral delivered genes to upregulate the production of neurotrophic factors in the inner ear to enhance nerve survival (Raphael). Important contributions to this work have also been made by Timo St�ver, a post-doctoral research fellow.

The purpose of this program of research has been to enhance the preservation of the auditory nerve following destruction of the sensory cells of the inner ear and to initiate a regeneration and regrowth of peripheral processes of the auditory nerve. Following loss of sensory cells of the inner ear, the peripheral processes of the auditory nerves die back to the cell bodies (spiral ganglion cells, SGC), and subsequently these cell bodies and proximal projections of the auditory nerve retreat into the brain stem, degenerate, and die. We know that the benefits of the cochlear prosthesis depend significantly upon the preservation of the auditory nerve and fibers, and there is strong evidence that there is an enhanced benefit from close contact between the stimulating electrodes and the auditory nerves and peripheral processes. Our work in this area began some years ago with the demonstration that chronic electrical stimulation (ES) following deafness could prevent the degeneration of the SGC. On the basis of this, we hypothesized that activity in the auditory nerve was a necessary survival factor for these cells and ES via prosthesis could provide an appropriate substitute for normal activity generated in these cells by the sensory cells of the inner ear, leading us to hypothesize we could obtain similar beneficial protective effects on the auditory nerve and peripheral processes with the application of neurotrophins.

Neurotrophins have been demonstrated as survival factors for neurons in vitro, particularly in the de-affected visual and auditory systems. In vivo, neurotrophins have been demonstrated as survival factors to enhance the survival of primary visual and auditory neurons following destruction of the receptor cells. In the auditory system, these findings may have direct clinical application for enhancement of the benefits of the cochlear implant for the profoundly deaf. This neural prosthesis functions by directly stimulating the auditory nerve, bypassing the damaged inner ear receptor. It is important from both a basic and a clinical perspective to demonstrate, in vivo, that enhanced survival of neurons is associated with enhanced responsiveness. We have now shown enhanced survival and/or regrowth of auditory neurons with multiple neurotrophins delivered alone or in combination. Importantly, we have shown improved function in neurotrophin-treated ears when we have used the auditory brainstem response to measure threshold sensitivity. The protective effects of neurotrophins have been evident with chronic infusion into the scala tymapni, as well as delivery using adenoviral gene therapy.

Previously, we performed studies with ES and observed that with the right intensity and frequency we were able to preserve SGCs. Based on this observation and results from other laboratories, we hypothesized 1) that the ES and activity of the auditory nerve up-regulates the expression of proteins that can provide long-term survival support for these cells, 2) that growth factors in particular may be up regulated by ES induced activity in these cells, and, 3) that this upregulation by ES may be dependent upon calcium channel pathways. We tested this hypothesis by delivering calcium channel blockers into the inner ear of chronically electrically stimulated animals to assess the change in auditory nerve survival. Collaborative work with other laboratories has demonstrated that depolarization of the auditory nerve in vitro results in increased cell survival. However, loss of Ca2+ to these cells is fatal, regardless of depolarization activity. With verapamil (a L-type Ca2+ channel blocker) chronically infused via an implantable mini-osmotic pump and microcannulation into the inner ear of chronically stimulated and unstimulated animals, we were able to reduce the protective effects of chronic stimulation on the auditory nerve cells without altering the evoked functional response of the ear. This supports our hypothesis that the protective effects of ES are dependent upon L-type calcium channels. This knowledge provides an important understanding of stimulation-induced nerve survival and makes an important advance in basic research, which will one day lead to new treatments with clinical application.

We have also investigated potential survival-enhancing effects of neurotrophin application on immature dorsal root ganglion (DRG) neurons and stem cells transplanted into the cochlea. Survival of DRG neurons and stem cells transplanted into the cochlea is enhanced by neurotrophic factors. In addition, we have found that stem cells differentiate into neurons, migrate to the auditory nerve, and project into the organ of Corti as well as the CNS. We have now identified these cells as immunoreactive to anti-glutamatergic antibodies.

Regeneration or replacement of the auditory nerve would provide a major new clinical intervention to reduce hearing loss. These results will thus have significant impact on the use of electrical stimulation and neurotrophins with and without viral vectors in future human clinical trials.

International Investigation

During the last decade a rich and active program of collaboration has developed between Dr. Miller's Tissue Engineering Laboratory at the KHRI, Dr. Colleen Le Prell (Univeristy of Florida), and laboratories in Sweden (Karolinska Institute and University of Uppsala), Finland (Turku University and University of Tampere), Germany (Hannover Medical University), Austria (University of Innsbruck), Australia (Melborne University), Japan (Kanzai University and Tokyo University) and Spain (Universidad de Castilla La Mancha). These collaborations include three main emphases:

  1. tissue engineering to promote auditory nerve regrowth following deafness
  2. prevention of noise-induced hearing loss
  3. improving the outcomes of cochlear prosthesis implantation (see BioEar)

These collaborations are facilitated by student fellowships and frequent investigator travel for targeted research projects.

1. Tissue engineering to promote auditory nerve regrowth following deafness

One model program reflecting these collaborations is focused on the translation of basic research observations on the mechanisms of cell survival and death following deafferentation to interventions that can be administered to promote auditory nerve survival and regrowth in the deaf patient receiving a cochlear implant. This program now involves a number of studies to bring our basic animal studies to the point of human clinical trials. These studies include:

  1. In vitro studies of human auditory nerves to evaluate responsiveness to neurotrophins found effective in animal studies and identification of precursor cells in the human temporal bone (Uppsala University; H. Rask-Andersen)
  2. Immunohistochemistry studies to identify the Trk receptors on human spiral ganglion cell that will be receptive to neurotrophin treatment (B)
  3. In-vivo animal studies to evaluate the effectiveness of delayed neurotrophin and antioxidant treatment on nerve survival, to better model the human situation (Karolinska Institute; M. Ulfendahl)
  4. In vivo animal investigation to evaluate the interactive effectiveness of antioxidants, electrical stimulation, and neurotrophins in promoting cell survival and regrowth of the auditory nerve (University of Michigan; J. Miller, R. Altschuler, Y. Raphael)
  5. In vivo animal studies to evaluate the interaction of electrical stimulation and neurotrophins on auditory nerve cell survival and the up-regulation of neurotrophins and their receptors in this tissue with deafness and treatment (Hannover Medical University; T. Stover, T. Lenarz)
  6. Development of human implants capable of short term chronic delivery of neurotrophins and antioxidants to the inner ear (Med-El Corp., Innsbruck)
  7. Toxicity studies of neurotrophins on the neural tissues of the inner ear (University of Michigan, J. Miller)

Together these studies provide the experimental rationale to permit preliminary human clinical trials in implanted patients.

2. Prevention of noise-induced hearing loss

A second major program with significant international cooperation is our program focused on the prevention of noise-induced hearing loss. These studies include:

  1. In vivo animal studies to evaluate the synergistic effects of antioxidants and other dietary supplements used to prevent noise induced hearing loss associated with exposure to chronic noise (University of Michigan; J. Miller, University of Florida; C. Le Prell; Washington University: K. Ohlemiller)
  2. In vivo animal studies to evaluate the synergistic effects of antioxidants and other dietary supplements used to prevent noise induced hearing loss associated with exposure to impulse noise (Karolinska Institute; M. Duan, G. Laurell)
  3. Human clinical trials to evaluate the effectiveness of antioxidants and other dietary supplements used to prevent noise-induced hearing loss as a consequence of impulse noise exposure during military training exercises (Karolinska Institute; U. Rosenhall, P.-A. Hellstrom, B. Hagerman, A.-C. Lindblad)
  4. Human clinical trials to evaluate the effectiveness of antioxidants and other dietary supplements used to prevent noise-induced hearing loss as a consequence of occupational noise exposure in NATO airbase personnel and workers in the cutlery industry(Universidad de Castilla La Mancha; J. Juiz)
  5. Human clinical trials for evaluation of the effectiveness of antioxidants and other dietary supplements in prevention of temporary noise-induced hearing loss as occurs with use of insert earphones and personal music players(University of Florida; C.Le Prell, J.Hall III; P. Antonelli)
  6. Toxicity studies of novel antioxidants, and novel combinations of antioxidants and other dietary supplements, on the neural tissues of the inner ear (University of Michigan; J. Miller)

Our US partners in all of the above human trials are Drs. Kathleen Campbell (Southern Illinois University School of Medicine) and Sharon Kujawa (Harvard University). Together, these studies will document the efficacy of a treatment to prevent noise-induced hearing loss in humans exposed to traumatic levels of sound as a consequence of recreational and/or occupational activities.

3. BioEar: Improving the outcomes of cochlear prosthesis implantation

Dr. Miller is the scientific director of an EU funded consortium working to improve the performance of the cochlear prosthesis. Thus, the studies described in this section reflect work performed not only in the Tissue Engineering Laboratory at the KHRI but also substantial efforts by an international consortium of academic and industrial partners. Academic partners include the Karolinska Institutet, Sweden, (Drs. Ulfendahl and Järlebark), University Of Hannover, Germany, (Drs. Lenarz, Stöver, and Reuter), University of Ferrara, Italy (Dr. Martini, and Maurizio Previati, MS), Uppsala University, Sweden (Dr. Rask-Andersen), the University of Insbruck, Austria (Dr. Schrott-Fischer), University of Tampere, Finland (Dr. Pyykkö) and Tampere University of Technology, Finland (Dr. Minna Kellomaki). Industrial partners include MED EL Elektromedizinische Geräte (Innsbruck, Austria), and BCI Bioabsorbable Concepts Ltd., (Tampere, Finland).

The cochlear implant (CI) has been the "success story" of neuroprosthetic devices. The development of this device, in the last 25 years, initially provided profoundly deaf patients with their first contact with the world of sound. With improvements in cochlear research technology have come progressive improvements in speech discrimination by implant users, and now substantial open-set (auditory only) speech discrimination achievable by the majority of implant patients. With continued development of signal processing strategies and electrode technology, the benefits to the deaf patient may yet increase, however it appears likely that present technology is optimized, and further progress limited by the status of the deprived auditory system itself.

There is still a large potential for improvement in cochlear implant outcomes. The major goal of this program is to improve the outcomes of cochlear implantation using drug application at the time of implantation. This could be achieved by (1) preventing neural degeneration during the traumatic procedure of implantation, and (2) initiating a re-growth of the peripheral processes of the auditory nerve towards the electrode array. Longer term goals are to:

  1. better understand the role of growth factors and other agents in preventing neural degeneration
  2. look towards the opportunity to provide these agents in patients with moderate hearing impairment to prevent its progress
  3. provide data that may make the use of these agents practical in other parts of the central nervous system (CNS)

The work therefore represents a scientific turning point, which has the potential to lead to immense future benefits for the community.

The immediate goal of the BioEar consortium is micro-fluidic drug-delivery in association with cochlear prosthesis implantation. Future generation drug delivery techniques will include biopolymer molecules coating the prosthesis and allowing slow delivery of drugs (such as growth factors or other agents), genes (for upregulating endogenous production of growth factors or other agents), or cells, into the deaf cochlea.

Technical Advances

Middle and Inner Ear Delivery Systems

Our laboratory has extensively used the micro-cannula and Alzet osmotic pump to deliver agents into the middle ear or directly into scala tympani of the guinea pig (Brown et al). Several changes to the delivery system have allowed for longer, more reliable infusion times (Prieskorn & Miller).

Electrode Stimulation System

Cytocochleograms

A computer program was developed in-house by Dr. David B. Moody to facilitate tabular and graphic presentation of cochlear sensory cell loss in guinea pig and mouse models. Microscope slide mounts of inner ear tissue are assessed for sensory cell loss, and missing cell counts are entered into the program. A graph (cytocochleogram) is generated depicting cell loss as a function of distance along the basilar membrane of the inner ear. This depiction is useful in various studies involving, e.g., noise exposure and drug infusions.