Sensory Hair Cell Survival & Death in the Inner Ear
The cochlea is the sensory organ responsible for hearing and plays a role in verbal communication. The sensory cells, or ‘hair cells’, of the organ of Corti are responsible for the transduction of acoustic input into nerve impulses that are then transmitted to the brain. Damage to or loss of hair cells leads to permanent hearing loss, as mammalian hair cells lack the ability to regenerate. According to the Center for Hearing and Communication, an estimated 38 million people in the United States—one of every ten Americans—have hearing loss, which imposes a huge detriment on the quality of life of the affected individuals and a formidable economic burden on society.
Our research focuses on acquired hearing loss, which is hearing loss that develops during one's lifetime secondary to exposure to influences noxious to the inner ear. Exposure to excessive noise or ototoxic drugs (e.g. aminoglycoside antibiotics or the anti-cancer agent cisplatin) and aging are the three main causes of acquired hearing loss. The histopathological hallmark of acquired hearing loss is the destruction of the hair cells in the inner ear, leading to a permanent loss of hearing.
The tools of our research include cell and molecular biology, immunohistochemistry, and physiology.
Noise-induced hearing loss (NIHL)
Research into NIHL using animal models has borne out two leading theories for the cause of hearing loss. One is mechanical damage from vibration of the organ of Corti beyond the tolerance of its physical structure. Another is a so called 'metabolic damage', wherein metabolic overstimulation and stress trigger cell death pathways. A variety of biochemical and molecular responses to noise trauma have been identified. For example, noise exposure elevates intracellular calcium levels in hair cells, likely through influx via calcium channels, and also activates calcineurin, a calcium-dependent phosphatase. Another well-documented response to noise trauma is the generation of reactive oxygen species (ROS). It appears that excessive noise decreases cochlear blood flow, which causes vasoactive lipid peroxidation and leads to generation of ROS. In addition, release of the neurotransmitter glutamate at inner hair cell synapses in response to traumatic noise has been implicated in excitotoxicity (neuronal damage and cell death from overstimulation by neurotransmitters) at this synapse.
The traditional method for prevention of NIHL by use of ear plugs to reduce noise levels reaching the ear has proven insufficient, primarily due to non-compliance. Based on animal experimentation, pharmacological prevention using a ‘hearing pill’ is a promising and appealing route. Antioxidants such as glutathione, D-methionine, ascorbic acid, and water soluble coenzyme Q10 have been reported to attenuate NIHL to some extent, while other antioxidants have failed to prevent NIHL. These conflicting results underscore the challenge still faced by the field in elucidating detailed molecular mechanisms and preventing NIHL.
(A) Auditory brainstem response (ABR) threshold shifts: The bar graph represents the permanent NIHL in 3-month-old male CBA/J mice resulting two weeks after exposure to 2–20 kHz broadband noise (BBN) at 106 dB SPL for two hours. Permanent threshold shifts (PTS) were greatest at high frequencies and decreased toward lower frequencies. Data are presented as means + SD; n = 5 for each condition.
(B) Images were taken with a 10× lens along the entire length of mouse cochlear epithelium from surface preparations prepared two weeks after exposure to 106 dB SPL 2–20 kHz BBN. No hair cells were missing in the apical region, while both outer and inner hair cells were completely destroyed in the basal region. An interesting pattern was observed in the middle segment of the cochlea. Hair cells were intact in the portion of the middle region nearest to the apex, while in the portion nearest to the base, outer hair cell loss ranged from partial to complete, although inner hair cells were still present.
(C) The images were taken with a 63× lens. The representative images illustrate hair cell losses in the middle region (3 mm from apex) two weeks after exposure to 106 dB SPL 2–20 kHz BBN. Preparations from control mice illustrate the normal structure of the sensory epithelium with three rows of outer hair cells (OHC), one row of inner hair cells (IHC) Scale bar = 10 µm.
PTS-noise exposure results in apoptotic and necrotic OHC nucleiBoth swollen necrotic (arrowheads), and condensed apoptotic nuclei (arrows) were present one hour post PTS-noise exposure. Red: propidium iodide staining of nuclei.
Noise trauma activates caspase-independent cell death pathways
Translocation of the mitochondrial protein endonuclease G (Endo G) into the nucleus indicates activation of caspase-independent cell death pathways. Immunofluorescent labeling of surface preparations from control mice showed Endo G in OHCs, but not in their nuclei. Three hours (3 h) post PTS-noise exposure, Endo G was translocated into condensed nuclei of some OHCs (arrows). Green: Endo G; blue: Hoechst 33342 staining for nuclei.
Noise trauma activates caspase-dependent cell death pathways
A strong active-caspase-3 signal was observed in OHCs three hours (3 h) after PTS-noise exposure. Images were taken from the upper basal turn. Green: cleaved caspase-3; red: propidium iodide staining of nuclei.
ATP-depletion agent oligomycin induces Endo G nuclear translocation in HEI-OC1 cells
Endo G staining appeared in the nuclei of some HEI-OC1 cells one hour after 1 µM oligomycin treatment (arrows). Endo G is stained green, and nuclei red with propidium iodide (PI).
Oligomycin induces HEI-OC1 cell death
Following incubation with 1 µM oligomycin for 24 hours, cell death of HEI-OC1 cells was detected by a flow cytometry assay. Oligomycin treatment significantly increased the amount of cell death compared to the control treatment. PI: propidium iodide.
Aminoglycoside-Induced & Age-Related Hearing Loss
Interestingly, noise-, and aminoglycoside-induced hearing loss and age-related hearing loss share similar pathologies and, at the very least, share oxidative stress as a pathological feature. We are, therefore, extending our studies to drug-induced and age-related hearing loss in collaboration with Dr. Jochen Schacht at the Kresge Hearing Research Institute at the University of Michigan. Both in-vivo and in-vitro studies from our laboratories have convincingly shown that oxidative stress is a causative factor in aminoglycoside-induced hearing loss and that this ototoxicity can be attenuated by the administration of antioxidants. We have collaborated with colleagues in the Department of Otolaryngology at Xijing Hospital in Xi'an, China, on a clinical study to protect against gentamicin-induced hearing loss using aspirin. Successful results showing a 75% reduction in the incidence of hearing loss were published in the New England Journal of Medicine in 2006. To date, this study shows the most striking protection reported against aminoglycoside-induced hearing loss and illustrates the principle that acquired hearing loss can be prevented with pharmacological interventions.
We have also collaborated in a program project grant studying the mechanism of age-related hearing loss (presbycusis) with other groups at the Kresge Hearing Research Institute and the Geriatrics Center at the University of Michigan. Our studies involve the assessment of free radical formation and cochlear antioxidant defenses as well as redox-sensitive homeostatic signaling pathways that engage protein kinases and phosphatases.
Current Personnel in the Sha Laboratory
Kayla Hill, BS, Graduate Student
Hong-Wei Zheng, M.D./Ph.D., Post-doc Research Fellow
Jun Chen, M.D./Ph.D., Post-doc Research Fellow
Hu Yuan, M.D./Ph.D., Post-doc Research Fellow
Yu Han, M.D./Ph.D., Post-doc Research Fellow
Past Personnel in the Sha laboratory
Carlene Brandon, MS, Research Specialist III
Fu-Quan Chen, M.D./Ph.D., Post-doc Research Fellow
Alison Kearns, MUSC Summer Undergraduate Student