Auditory Biochemistry and Molecular Biology Laboratory
Research
Quick Jump:
- A Brief Introduction to the Inner Ear
- Regulatory Mechanisms in the Inner Ear
- Drug-induced Hearing Loss
- Age-related Hearing Loss
A Brief Introduction to the Inner Ear
The inner ear is a tiny but complex structure that is firmly embedded in the skull bone. The two major parts of the inner ear contain the tissues responsible for our sense of hearing (cochlea) and balance (vestibular organ). The sounds that we perceive from the environment are vibrations of air molecules reaching our ear. They travel through the ear canal and impinge on the eardrum. There they set the small bones of the middle ear (ossicles) in motion, which in turn vibrate the fluids in the inner ear. The sound waves then travel through the inner ear, like waves that travel in water when a rock is thrown. The special sensory cells in the inner ear (hair cells) pick up these vibrations and transduce them to information that is carried to the brain by the auditory nerve.
What the Inner Ear Looks Like
The hair cells that process acoustic information are part of the "organ of Corti".
A view of the structural organization of this organ
as seen through a scanning electron microscope.
The white tufts in the above picture are the sensory hairs (stereocilia) on the top of each cell � hence the name "hair cell". The upper row represents the hairs of the "inner hair cells" and the three rows of w-shaped hairs indicate the position of the "outer hair cells". The cell bodies of the outer hair cells extend to the bottom of the picture. The diameter of the cell body of an outer hair cell is 10 micrometers, less than 1/1000th of an inch. In between the sensory cells we see the so-called "supporting cells".
The hair cells are very sensitive structures. When hearing loss is caused by noise or drugs we frequently find that these hair cells are destroyed. Since they cannot regenerate themselves, the resulting hearing loss is permanent.
Damaged organ of Corti
The picture above shows an organ of Corti damaged after exposure to loud noise. The loss of outer hair cells is quite evident by the disappearance of the sensory hairs, for example as indicated by the arrow.
Some Ways to Study the Biochemisty of Inner Ear Tissues
Biochemical and cell biological investigations use a number of different techniques to investigate these tiny tissues. It is often said that biochemists like to "grind up" tissues for analysis, and it is indeed one of our approaches to use such tissue homogenates. However, it is also necessary and possible to study biochemical reactions in intact cells or tissue preparations.
Immunocytochemistry is a technique in which specific cell components are labeled with "antibodies" so that we can determine their localization in a tissue or cell. The antibodies can then be detected under the microscope by their color.
Prepared organ of Corti that is stained with an antibody
specifically recognizing the supporting cells
The picture above shows a preparation of the organ of Corti that is stained with an antibody specifically recognizing the supporting cells. If we compare this picture to the first photo, it becomes apparent that the positions of hair cells are marked by "black holes", while the supporting cells in between the hair cells fluoresce in red.
Modern technology (laser scanning confocal microscopy) and the development of sophisticated analytical methods make it possible to visualize an ever increasing number of cellular components.
Cross-section of an organ of Corti
The photo above is an example and an esthetically pleasing picture as well. In this cross-section of the organ of Corti the expert would recognize the outer hair cells (to the right), the inner hair cell (to the left of the "tunnel") as well as supporting cells. The red dye signals the products of an attack of free radicals on cell lipids; the green dye traces the action of nitric oxide; and in the yellow areas, both reactions are happening. In addition, the nuclei of the cells are stained blue which makes it easy to read the morphology and recognize the cell types.
Investigations of Individual Isolated Sensory Cells
Much work in our laboratory has focused on studies of isolated outer cells of the guinea pig. The cigar-like shape of the outer hair cells is nicely seen in the photo below.
Outer hair cells
Close inspection of the cells reveals the tufts of hair, stereocilia, at the upper end of some of the individual hair cells. In these preparations, we have studied biochemical mechanisms such as the motility of hair cells, "second messengers", and the regulation of intracellular calcium ions (for details, please, see regulatory mechanisms in the inner ear).
A valuable tool for such studies are fluorescent indicators which are specific reporters for certain cellular components. The photo below shows the visualization of calcium ions in an isolated hair cell.
Visualization of calcium ions in an isolated hair cell
The four images show the same cell and represent a timed sequence of changes in its calcium content. The fluorescent images are converted by the computer to "false colors" indicating the intensity of the fluorescence (scale at the bottom).
The pictures above were taken by Hong-Yan Jiang and Gary Zajic.
Regulatory Mechanisms in the Inner Ear
As in all organs of our bodies, the many cells in the inner ear must cooperate in order to do their job of processing acoustic information so that we may understand our environment. Each cell has a special task in this concerted effort. In order to communicate with each other and with the environment, cells send out and receive signals. For example, signals from the environment may come in the form of sensory information and signals from other cells may come in the form of hormones. These extracellular signals change intracellular metabolism but, with few exceptions, never enter the cell. This apparent paradox was first resolved in the 1970's when the concept of "second messengers" was discovered. External signals bind to specific receptors in the membrane surrounding each cell. Here they trigger the generation of intracellular second messengers which mediate the "first message" of the external signals. The second messengers then control and adjust the cellular physiology and metabolism.
Second messengers include compounds such as cyclic AMP, cyclic GMP, inositol trisphosphate, and calcium. They exert their effects by changing the activity of enzymes or other cellular constituents. This frequently happens through modification of proteins by a phosphorylation reaction mediated by protein kinases. The processing of an external signal thus is a rather complex cascade of the first messenger acting at the cell membrane, followed by the generation of second messengers, the activation of protein kinases, and the modification of proteins.
Second messengers and protein kinases are widely distributed in the cochlea. Our studies have focused on second messenger mechanisms involving nitric oxide/cyclic GMP pathway, inositol trisphosphate, calcium and protein kinases in an attempt to elucidate the processes that control the function of the inner ear. Our current studies focus on those signaling pathways that control the survival or death of our hair cells. In particular, we are looking at pathways that are mediated by phospoinositide-3-kinase, Akt, or NF-kB. Small Rho GTPases apparently play a crucial role in the control of the actin cytoskeleton which is essential in the maintenance of the structural integrity of the organ of Corti. NF-kb and the redox-regulated signaling pathways related to this transcription factor appear to constitute a survival mechanism in the inner ear. Other pathways of interest for us are those that control cell death via apoptosis or necrosis. These studies of cell signaling mechanisms strongly overlap withour studies on drug- and noise-induced hearing loss since we are frequently using aminoglycoside antibiotics and noise trauma as tools to investigate these pathways.
See also recent publications on regulatory mechanisms in the inner ear.
Drug-induced Hearing Loss
In spite of efforts to develop new and better medication, several classes of common and thus far indispensable drugs can harm the cells in the inner ear and thereby affect our hearing or balance. These drugs are referred to as "ototoxic". Ototoxic drugs belong to such diverse therapeutic classes as antibacterial agents, diuretics, antimalarials, anticancer agents, and even non-prescription pain killers (e.g., aspirin). They can produce either temporary or permanent symptoms such as tinnitus (ringing in the ear), hearing loss, or vestibular dysfunction (dizziness, loss of balance). Aspirin in high doses, for example, may lead to ringing in the ears and some loss of hearing sensitivity. Fortunately, its effects are only temporary and hearing will return to normal when aspirin is no longer taken. Medications that have the potential to cause permanent damage are only used under careful clinical supervision in most countries.
Although we have developed models for the study and prevention of cisplatin - induced hearing loss, our research has focused on aminoglycoside antibiotics. This class of drugs includes amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, and tobramycin all of which are highly effective against bacterial infections. Unfortunately, they can cause permanent loss of hearing or balance. In the United States, aminoglycosides are mostly used for emergency treatment of people with serious infections who have not responded to other types of antibiotics. Worldwide, however, they are the most commonly used antibiotics due to their high effectiveness and low cost. They too frequently are the only affordable medication in less affluent and more populous countries of southeast Asia, the Indian subcontinent, Africa and South America. Studies from some of these countries report a staggering incidence of side-effects with perhaps more than 100,000 people affected annually.
Our laboratory has recently developed a theory that allows us both to understand aminoglycoside toxicity and prevent it. Aminoglycosides can bind to a small portion of the body's iron in a very specific way. By doing so, they form a drug/iron complex that can generate "free radicals". Free radicals are a group of highly aggressive compounds that have the potential of leading to tissue injury and cell death. In fact, free radicals are known or suspected as the causative agents in a great number of diseases or disorders. Our current studies focus on the signaling pathways that are triggered in the cell in response to a change in the "redox status" by the action of free radicals. Depending on the intensity of the stimulus, in this case the dose and duration of aminoglycoside treatment, these pathways can lead to survival or to cell death. in this context we have identified redox regulated and NF-kB medicated pathways as a survival mechanism in cells of the inner ear. In contrast, cell death in vivo mostly eappears to involve caspase-independent necrotic or necrotic-like mechanisms medicated, for example, by calpains and cathepsins. We supplement the in-vivo studies by experiments in specific cell lines that faciliate the elucidation of such complex mechanisms.
Based on the results of our work on the basic mechanisms, we have been developing preventive treatments of the side effects of aminoglycosides. This is accomplished by administration of �antioxidants� that neutralize the harmful radicals and steer cell pathways towards survival. Indeed, administration of antioxidants drastically reduced gentamicin ototoxicity in guinea pigs and mice. The effectiveness of this "antioxidant therapy" is not limited to gentamicin, but also extends to other aminoglycoside antibiotics and, importantly, does not impair the antibacterial activity of aminoglycosides. We explored the potential for clinical practice in collaboration with our colleagues at the Xijing Hospital, Fourth Military Medical University in Xi�an, China. The protective therapy consisted of aspirin given concomitantly and the results exceeded our expectations, showing a 75% reduction in risk of hearing loss. The study was published in the New England Journal of Medicine and you can check out this University of Michigan press release.
In addition, check out this University of Michigan press release about our not-so-recent results.
Last but not least, see our recent publications on drug-induced hearing loss.
Age-related Hearing Loss
Age-related hearing loss, or presbycusis, is a major health concern. Approximately 44% of people suffer from a significant hearing loss by age 69. This number rises to 66% by age 79, and skyrockets to 90% after age 80. As a consequence, communication with the elderly is severely impaired, leading to social isolation and loss in quality of life. As the average age of our population steadily increases, the magnitude of problem increases.
Age-related hearing loss usually involves a progressive decrease in the auditory system's sensitivity to higher frequency sounds, as measured by an elevation of our hearing thresholds. Presbycusis is clearly multifaceted and a variety of pathologies associated with age-related hearing loss have been described over the years, suggesting that alterations in many cell types may be responsible for sensory loss. In the cochlea, prominent pathologies include loss of sensory hair cells, often in the basal region of the cochlea that is most sensitive to high frequency sounds. But there is also degeneration of the strial region in the lateral wall of the cochlea that mediates ionic balance in the ear, and loss of spiral ganglion cells of the auditory nerve.
In the course of a lifetime of sixty years or more, the human ear is exposed to such a variety of environmental insults (e.g., noise, ototoxic agents), diet, cardiovascular or other disease conditions, that "pure" presbyacusis may be merely a hypothetical concept, never to be found in the real world of human otology. It should be possible to observe an unadulterated presbyacusis in noise-protected laboratory animals, but their pattern of presbyacusis is also somewhat variable. Aging mice, rats and gerbils are models that reflect various aspects of human prebycusic changes. Our choice of animal is the mouse, primarily based on the availability of molecular and genetic information and of transgenic and knock-out animals.
The major hypothesis underlying our studies is derived from our general knowledge of the aging process. One theme that has emerged recently is the relationship between tissue survival and increased resistance to oxidant stress. Genetic investigations initially in invertebrates but later also in mammals have shown intimate links between lifespan and tissue function and stress resistance. Mutations in the nematode C. elegans leading to lifespan extension typically also lead to resistance to multiple forms of stress including oxidant stress.
Our project - which was developed by Dr. Sha (Shasha) - hypothesizes that oxidant stress also contributes to age-related hearing loss. Specific studies involve the determination of the cochlear antioxidant defenses s a first approximation of oxidant stress. Secondly, we are studying redox-sensitive homeostatic signaling pathways involving protein kinases and phosphatases, leading to the activation of transcription factors and culminating in gene transcription to increase protective and antioxidant enzymes. Finally, cell death pathways are part of our program since ultimately our hair cells will succumb to the stress of aging.
