The principal aims of the studies described in this thesis were to
develop an in vitro model for studying acoustic overstimulation at the
cellular level, to define the electrical and mechanical response
characteristics of the perfused temporal bone preparation, and to
investigate the effects of intense sound stimulation on the calcium
levels of the hair cells in the intact hearing organ. In the in vitro
model for acoustic overstimulation, isolated cochlear outer hair cells
were subjected to a pressure jet emanating from a glass micropipette
aimed at the cell body. The pressure jet was generated by the vibrating
shaft of a minishaker, hydraulically coupled to the micropipette. Such
stimulation was found to cause increases of the cytoplasmic calcium
concentration in most auditory sensory cells. The calcium changes were
sustained, and no evidence of recovery of the elevated levels were seen
after the termination of the stimulus. A system was also developed to
measure the pressures delivered to the cells. This system was based on a
piezoresistive pressure transducer connected to a glass micropipette
brought into the immediate vicinity of cells subjected to the pressure
jet, allowing highly localized pressure changes to be measured. The peak
pressure that could be generated by the stimulus system was found to be
144 dB SPL. Taking the middle ear transfer function into account, this
level would correspond to approximately 120 dB SPL at the tympanic
membrane during normal sound stimulation. Using laser heterodyne
interferometry, the sound-induced vibrations of the low-frequency regions
of the inner ear were investigated. The responses of the perfused
isolated temporal bone preparation was found to be similar to that of
living animals, both in terms of sharpness of tuning and the presence of
nonlinearities. These characteristics were also reflected in the
extracellularly recorded receptor potentials of the hair cells. The
mechanical and electrical responses of the low-frequency regions of the
cochlea were substantially different from the high-frequency regions,
however. Methods were developed to load the organ of Corti with
fluorescent dyes and to measure the fluorescence after various
experimental manipulations, using video-enhanced microscopy. The
fluorescence images were further processed off-line, using a computerized
algorithm, to remove out-of-focus information. When the isolated temporal
bone preparation was subjected to acoustic overstimulation, large
increases of the calcium concentration of the outer hair cells were seen.
In addition, overstimulation caused contractions of the hearing organ
that were reversible after the termination of the stimulus. Both the
calcium changes and the contraction response could be expected to have
severe effects on the function of the inner ear.
Keywords: Cochlea, outer hair cells, calcium, pressure changes,
micromechanics, noise-induced hearing loss, fluorescent indicators, laser
interferometry, temporal bone preparation, guinea pig
ISBN 91-628-2353-
