Increased vitamin plasma levels in Swedish military personnel treated with nutrients prior to automatic weapon training.

Noise-induced hearing loss (NIHL) is a significant clinical, social, and economic issue. The development of novel therapeutic agents to reduce NIHL will potentially benefit multiple very large noise-exposed populations. Oxidative stress has been identified as a significant contributor to noise-induced sensory cell death and NIHL, and several antioxidant strategies have now been suggested for potential translation to human subjects. One such strategy is a combination of beta-carotene, vitamins C and E, and magnesium, which has shown promise for protection against NIHL in rodent models, and is being evaluated in a series of international human clinical trials using temporary (military gunfire, audio player use) and permanent (stamping factory, military airbase) threshold shift models (NCT00808470). The noise exposures used in the recently completed Swedish military gunfire study described in this report did not, on average, result in measurable changes in auditory function using conventional pure-tone thresholds and distortion product otoacoustic emission (DPOAE) amplitudes as metrics. However, analysis of the plasma samples confirmed significant elevations in the bloodstream 2 hours after oral consumption of active clinical supplies, indicating the dose is realistic. The plasma outcomes are encouraging, but clinical acceptance of any novel therapeutic critically depends on demonstration that the agent reduces noise-induced threshold shift in randomized, placebo-controlled, prospective human clinical trials. Although this noise insult did not induce hearing loss, the trial design and study protocol can be applied to other populations exposed to different noise insults.

In vitro studies of cochlear excitation.

Although initially met with scepticism, the in vitro temporal bone preparation of the cochlea has proved to be a very important tool for investigating the function of the mammalian auditory system. As present techniques are able to maintain sufficient cellular viability, the in vitro preparation offers a valuable bridge between investigations using isolated outer hair cells and the intact system in vivo.

Differential gene expression in the rat cochlea after exposure to impulse noise.

Understanding the molecular biology of noise trauma is vital to developing effective and timely interventions. In a model of explosion-mediated impulse noise injury, differential gene expression was studied in whole rat cochlea preparations at 3 and 24 h following the exposure. We developed a technique using mRNA from a single cochlea on each oligonucleotide microarray to avoid pooling of mRNA samples. Application of a conservative statistical analysis approach resulted in the identification of 61 differentially expressed genes. Within 3 h after the exposure, there was an up-regulation of immediate early genes, mainly transcription factors and genes involved in the tissue's response to oxidative stress. No genes were found to be significantly down-regulated. At 24 h following the exposure, up-regulated genes included members of inflammatory and antioxidant pathways and one gene involved in glutathione metabolism was down-regulated. A subset of genes was confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The present study demonstrates the power of the microarray technique in providing a global view of the gene regulation following noise exposure, and in identifying genes that may be mechanistically important in hearing loss, and thereby serve as a basis for the development of therapeutic interventions.

Image adaptive point-spread function estimation and deconvolution for in vivo confocal microscopy.

Visualizing deep inside the tissue of a thick biological sample often poses severe constraints on image conditions. Standard restoration techniques (denoising and deconvolution) can then be very useful, allowing one to increase the signal-to-noise ratio and the resolution of the images. In this paper, we consider the problem of obtaining a good determination of the point-spread function (PSF) of a confocal microscope, a prerequisite for applying deconvolution to three-dimensional image stacks acquired with this system. Because of scattering and optical distortion induced by the sample, the PSF has to be acquired anew for each experiment. To tackle this problem, we used a screening approach to estimate the PSF adaptively and automatically from the images. Small PSF-like structures were detected in the images, and a theoretical PSF model reshaped to match the geometric characteristics of these structures. We used numerical experiments to quantify the sensitivity of our detection method, and we demonstrated its usefulness by deconvolving images of the hearing organ acquired in vitro and in vivo.

Mechanical responses of the mammalian cochlea.

Recent findings in auditory research have significantly changed our views of the processes involved in hearing. Novel techniques and new approaches to investigate the mammalian cochlea have expanded our knowledge about the mechanical events occurring at physiologically relevant stimulus intensities. Experiments performed in the apical, low-frequency regions demonstrate that although there is a change in the mechanical responses along the cochlea, the fundamental characteristics are similar across the frequency range. The mechanical responses to sound stimulation exhibit tuning properties comparable to those measured intracellularly or from nerve fibres. Non-linearities in the mechanical responses have now clearly been observed at all cochlear locations. The mechanics of the cochlea are vulnerable, and dramatic changes are seen especially when the sensory hair cells are affected, for example, following acoustic overstimulation or exposure to ototoxic compounds such as furosemide. The results suggest that there is a sharply tuned and vulnerable response related to the hair cells, superimposed on a more robust, broadly tuned response. Studies of the micromechanical behaviour down to the cellular level have demonstrated significant differences radially across the hearing organ and have provided new information on the important mechanical interactions with the tectorial membrane. There is now ample evidence of reverse transduction in the auditory periphery, i.e. the cochlea does not only receive and detect mechanical stimuli but can itself produce mechanical motion. Hence, it has been shown that electrical stimulation elicits motion within the cochlea very similar to that evoked by sound. In addition, the presence of acoustically-evoked displacements of the hearing organ have now been demonstrated by several laboratories.

Supporting cells contribute to control of hearing sensitivity.

The mammalian hearing organ, the organ of Corti, was studied in an in vitro preparation of the guinea pig temporal bone. As in vivo, the hearing organ responded with an electrical potential, the cochlear microphonic potential, when stimulated with a test tone. After exposure to intense sound, the response to the test tone was reduced. The electrical response either recovered within 10-20 min or remained permanently reduced, thus corresponding to a temporary or sustained loss of sensitivity. Using laser scanning confocal microscopy, stimulus-induced changes of the cellular structure of the hearing organ were simultaneously studied. The cells in the organ were labeled with two fluorescent probes, a membrane dye and a cytoplasm dye, showing enzymatic activity in living cells. Confocal microscopy images were collected and compared before and after intense sound exposure. The results were as follows. (1) The organ of Corti could be divided into two different structural entities in terms of their susceptibility to damage: an inner, structurally stable region comprised of the inner hair cell with its supporting cells and the inner and outer pillar cells; and an outer region that exhibited dynamic structural changes and consisted of the outer hair cells and the third Deiters' cell with its attached Hensen's cells. (2) Exposure to intense sound caused the Deiters' cells and Hensen's cells to move in toward the center of the cochlear turn. (3) This event coincided with a reduced sensitivity to the test tone (i.e., reduced cochlear microphonic potential). (4) The displacement and sensitivity loss could be reversible. It is concluded that these observations have relevance for understanding the mechanisms behind hearing loss after noise exposure and that the supporting cells take an active part in protection against trauma during high-intensity sound exposure.

The cellular localization of the neuropeptides substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y in guinea-pig vestibular sensory organs: a high-resolution confocal microscopy study.

Four neuropeptides, substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y, were detected by radioimmunoassay in guinea-pig vestibular end-organs. High-resolution confocal microscopy visualization of immunofluorescence staining was used to determine the cellular localization of these peptides. Substance P- and neurokinin A-like immunoreactivities were found to co-exist in afferent fibers innervating the peripheral regions of both the utricular and ampullar sensory organs. The immunoreactivity was more concentrated in the distal ends of the calyceal-shaped nerve endings that innervate type I sensory cells. While in the guinea-pig, nerve calyces and type I cells are distributed in both the central and peripheral regions of the sensory epithelia, immunoreactive calyces were found only in the peripheral regions. Calcitonin gene-related peptide-like immunoreactivity was localized in small bouton endings situated at the level of the base of the hair cells. These boutons were in a position to make axosomatic contacts with type II sensory cells and axodendritic contacts with afferent nerve endings. Calcitonin gene-related peptide immunoreactivity co-existed with choline acetyltransferase immunoreactivity. The localization and shape of these boutons identified them as the axonal endings of efferent vestibular fibers. Neuropeptide Y-like immunoreactivity was not observed in the actual sensory epithelium but in the underlying connective tissue, where it was located in varicose fibers along blood vessels. The synaptic position of the tachykinins is clearly distinct from that of calcitonin gene-related peptide. This segregation distinguishes the vestibular end-organs from most peripheral tissues where these peptides are co-localized. The tachykinin-immunoreactive afferent fibers are postsynaptic to the hair cells. If, as in somatic sensory endings, these fibers can be triggered to release the neuropeptides by an axon reflex type of activation, then the tachykinins could interfere directly with the function of type I and type II vestibular hair cells. Calcitonin gene-related peptide co-exists with acetylcholine in the efferent axonal endings that are presynaptic to type II hair cells and to afferent fibers. Calcitonin gene-related peptide can thus interfere by direct synaptic action with type II hair cells only. It may also regulate the activity of the tachykinin-containing afferents.

Vital staining of the hearing organ: visualization of cellular structure with confocal microscopy.

Cells inside the intact organ of Corti were labelled with fluorescent probes reflecting various aspects of structure and function. The dyes were introduced into the perilymphatic space by perfusion of the scala tympani of the temporal bone from the guinea-pig maintained in isolation. The dyes were able to diffuse through the basilar membrane and into the organ of Corti where they were spontaneously absorbed by the sensory and supporting cells. Confocal microscopic observation was made through an opening in the apex of the cochlea. A number of different dyes were used; a carbocyanine dye which stains mitochondria; two styryl dyes which are absorbed by the cell membranes and calcein, a cytoplasmic marker that fluoresces in vital cells. Extracellular space was stained by a cell-impermeant Dextran fluorescein. The most striking finding was that the membrane dyes preferentially stained the sensory cells and neural elements whereas the staining of the supporting cells was faint. The cytoplasmic dye in general stained sensory and supporting cells to the same extent. By tilting the organ, a view could be obtained from the side like a radial section through the organ. Outer and inner hair cells with their sensory hairs, nerve fibres and nerve endings, especially under the inner hair cells, could be seen in profile. Introduction of a high molecular weight Dextran into the endolymphatic space outlined the tectorial membrane which was seen in negative contrast. The simultaneous perfusion with a membrane dye stained the hair cells and their sensory hairs. Merging of the two images gave the possibility to examine, in the living tissue, the cilia to tectorial membrane relationship. Of general interest is the finding that the membrane dyes preferentially stained the sensory and neural elements of the nervous system, represented here by the hair cells and nerve fibres of the inner ear.

Acoustic trauma causes reversible stiffness changes in auditory sensory cells.

A common cause of hearing impairment is exposure to loud noise. Recent research has demonstrated that the auditory mechanosensory cells are essential for normal hearing sensitivity and frequency selectivity. However, little is known about the effect of noise exposure on the mechanical properties of the auditory sensory cells. Here we report a significant reduction in the stiffness and cell length of the outer hair cells after impulse noise exposure, suggesting that mechanical changes at the cellular level are involved in noise-induced hearing loss. There is a recovery of the cellular stiffness and cell length over a two-week period, indicating an activation of cellular repair mechanisms for restoring the auditory function following noise trauma. The reduced stiffness observed at the cellular level is likely to be the cause for the downward shift of the characteristic frequency seen following acoustic trauma. The deterioration and the recovery of the mechanical properties of outer hair cells may form important underlying factors in all kinds of noise-induced hearing loss.

The vibration pattern of the hearing organ in the waltzing guinea-pig measured using laser heterodyne interferometry.

The mechanical tuning characteristics of the hearing organ were measured in response to sound stimulation using laser heterodyne interferometry in in vitro preparations of temporal bones from waltzing guinea-pigs expressing different degrees of hearing organ and sensory cell degeneration. Measurements were made at various stages of structural changes allowing us to correlate structure and mechanical function. It was found that the characteristic frequency of the response at a given location in the cochlea occurred at lower frequencies than what is normally seen and that the sharpness of the mechanical tuning was considerably reduced when sensory hair cells were absent and the hearing organ structurally altered. However, even when extensive hair cell degeneration was evident a residual mechanical tuning was present. These results further support the concept that the sensory hair cells plays a key role in determining normal auditory tuning characteristics. It is suggested that the basilar membrane mechanics gives rise to a broadly tuned mechanical response on which a sharper tuning mechanism, originating from the hair cells, is superimposed.

Early compensation of vestibulo-oculomotor symptoms after unilateral vestibular loss in rats is related to GABA(B) receptor function.

The horizontal vestibulo-oculomotor reflex was studied in pigmented rats during the first 5 days after a unilateral chemical or surgical vestibular deafferentation. Spontaneous eye movements in darkness and slow phase velocity gain of compensatory eye movements during horizontal sinusoidal rotation were evaluated. The most evident vestibulo-oculomotor symptom immediately after a unilateral vestibular loss was a spontaneous nystagmus, which gradually abated during the following days. Further, an asymmetry between ipsi- and contra-lesional gains was evident during sinusoidal vestibular stimulation. Single systemic doses of the GABA(B) receptor antagonist [3-[1-(S)-[[3-(cyclohexylmethyl)-hydroxyphosphinoyl]-2-(S)-hydroxypropyl]amino]ethyl]-benzoic acid (CGP 56433A), the agonist baclofen, or the GABA(A) receptor agonist (4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol (THIP) were given at different intervals after unilateral vestibular deafferentation. CGP 56433A highly aggravated the vestibulo-oculomotor symptoms, observed as an increase in spontaneous nystagmus and slow phase velocity gain asymmetry. This effect was most pronounced during the first 2 days after unilateral vestibular loss, when CGP 56433A even decompensated the vestibular system to the extent that all vestibular responses were abolished. Baclofen caused no effect during the first days after unilateral vestibular loss, but in parallel with the abatement of spontaneous nystagmus, the drug equilibrated or even reversed the remaining spontaneous nystagmus with corresponding effects on the slow-phase velocity gain asymmetry. The effects of baclofen were very similar after both chemical and surgical deafferentation. THIP caused a slight depression of all vestibular responses. All single dose effects of the drugs were transient. Altogether these results reveal that endogenous stimulation of GABA(B) receptors in GABA-ergic vestibulo-oculomotor circuits are important for reducing the vestibular asymmetry during the early period after unilateral vestibular deafferentation. A possible role for GABA(B) receptors in the reciprocal inhibitory commissural pathways in the vestibular nuclei is suggested.

The tuned displacement response of the hearing organ is generated by the outer hair cells.

The motile responses of the guinea-pig hearing organ in response to a tone applied to the ear were measured by laser interferometry. Two types of responses can be recorded: (i) a vibration at the frequency of the applied tone; and (ii) a displacement response consisting of a shift in the position of the organ surface. The purpose of this study is to characterize the displacement response. The results are as follows. There is a relationship between the frequency of highest sensitivity (best-frequency) of the displacement response and the site from which it is recorded. High best-frequencies are noticed at more basal locations, low best-frequencies towards the apex. The displacement response is more frequency-selective than the vibration response. The displacement response is observed within physiological sound pressure levels. Its sharpness is dependent on the stimulus intensity, it shows biological variability and can be manipulated by drugs that are known to modify the receptor potential of the sensory cells, or to interfere with outer hair cell motility. These results suggest that the displacement response is an important step in the transduction process in the mammalian hearing organ and that it is generated by the motile action of the outer hair cells.

Shearing motion in the hearing organ measured by confocal laser heterodyne interferometry.

To investigate the presence of the postulated shearing motion in the micromechanics of the inner ear during sound stimulations we measured the vibratory response of the tectorial membrane and the reticular lamina in the third cochlear turn in an isolated temporal bone preparation using confocal laser heterodyne interferometry. The mechanical response of the tectorial membrane had the same frequency of maxima as the underlying reticular lamina, but was not as sharply tuned. When the two-dimensional motion was calculated from measurements made from several viewing angles it was found that the vibration of the reticular lamina had significant components both normal and tangential to its surface. The tectorial membrane motion, however, was primarily in a direction approximately perpendicular to the surface of the reticular lamina. The results indicate that shearing motion is produced predominantly by the radial motion of the reticular lamina.

Frequency-specific position shift in the guinea pig organ of Corti.

The organ of hearing is tuned as expressed both in the vibratory response of the cochlear partition and in the resulting receptor potentials of the sensory cells. We now demonstrate a sharply tuned response, consisting of a position shift of the surface of the organ of Corti, occurring during the presentation of a tone. The magnitude of the position shift exceeds that of the vibratory response to the stimulus. The shift is most pronounced in the region of the outer hair cells, and its affected by an inhibitor of outer hair cell motility. We conclude that the response is induced by the action of the outer hair cells.

Mechanically evoked shortening of outer hair cells isolated from the guinea pig organ of Corti.

Outer hair cells isolated from the mammalian hearing organ have been shown to respond to mechanical stimuli at acoustic frequencies by expressing a change in cell length (e.g. Canlon et al., 1988). The acoustically evoked response is characterised by both a tonic length change following the envelope of the stimulus, and a frequency-dependent phasic component. We show here that mechanical stimulation at much lower frequencies directed at the cell body also elicits length changes of the outer hair cells. When the apical pole of isolated outer hair cells was compressed with a quartz fibre, a shortening or contraction at the basal pole was observed. Transverse indentation at the lateral membrane elicited shortenings at both ends of the cells. The sensitivity to the mechanical manipulation was changed by an altered tonicity of the external solution, or exposure to salicylate. As the response occurs at very low stimulus frequencies, it may account for the mechanism by which the hearing organ responds to the low frequency modulation component in complex signals like speech.

Shortening and elongation of isolated outer hair cells in response to application of potassium gluconate, acetylcholine and cationized ferritin.

Individual outer hair cells isolated from guinea pig cochleae were observed in vitro during the application of solutions that are known to cause hair cells to shorten. Solutions containing high potassium, which depolarizes cells, were applied in the form of potassium gluconate. The initial response was a shortening, followed by an elongation, after which the hair cells nearly resumed their original length. Solutions containing the presumed efferent neurotransmitter acetylcholine also caused an initial shortening, occasionally followed by an elongation, where a cell either returned to normal or exceeded its original length. Solutions containing cationized ferritin caused some cells to shorten and caused others to lengthen. The results indicate that the hair cell response to a chemical stimulus can be bidirectional. Moreover, the initial response of an individual cell may depend not only on the stimulus but also on the physiological state of the hair cell or the original location of the hair cell along the length of the sensory epithelium when it was in the cochlea.

Effects of caffeine and tetracaine on outer hair cell shortening suggest intracellular calcium involvement.

Outer hair cell (OHC) shortening has previously been induced in vitro by the application of solutions containing high potassium (a depolarizing agent), acetylcholine (a suggested efferent transmitter) and cationized ferritin (a positively charged macromolecule), as well as by electrical current. The application of caffeine, which causes contractures in skeletal and smooth muscle by releasing calcium from intracellular stores to activate actin and myosin interaction, also causes shortening of OHCs. Tetracaine, which interferes with calcium movement in muscle and non-muscle cells, blocks potassium-induced and caffeine-induced shortening of OHCs, but does not block electrically-induced shortening. Sodium dantrolene which is an inhibitor of intracellular calcium release in skeletal muscle does not block potassium-induced OHC shortening. Immunocytochemical studies using antibodies to muscle-like contractile and regulatory proteins on unfixed, freeze-dried OHCs demonstrate the co-localization of calmodulin with actin throughout the OHC cytoplasm. These results support the ideas that in OHCs, intracellular calcium release is involved in the activation of shortening and that an actin-mediated cell shape change may be regulated by calmodulin in a manner similar to that which occurs in contraction of smooth muscle.

Actin-binding and microtubule-associated proteins in the organ of Corti.

Actin-binding and microtubule-associated proteins regulate microfilament and microtubule number, length, organization and location in cells. In freeze-dried preparations of the guinea pig cochlea, both actin and tubulin are found in the sensory and supporting cells of the organ of Corti. Fodrin (brain spectrin) co-localized with actin in the cuticular plates of both inner and outer hair cells and along the lateral wall of the outer hair cells. Alpha-actinin co-localized with actin in the cuticular plates of the hair cells and in the head and foot plates of the supporting cells. It was also found in the junctional regions between hair cells and supporting cells. Profilin co-localized with actin in the cuticular plates of the sensory hair cells. Myosin was detected only in the cuticular plates of the outer hair cells and in the supporting cells in the region facing endolymph. Gelsolin was found in the region of the nerve fibers. Tubulin is found in microtubules in all cells of the organ of Corti. In supporting cells, microtubules are bundled together with actin microfilaments and tropomyosin, as well as being present as individual microtubules arranged in networks. An intensely stained network of microtubules is found in both outer and inner sensory hair cells. The microtubules in the outer hair cells appear to course throughout the entire length of the cells, and based on their staining with antibodies to the tyrosinated form of tubulin they appear to be more dynamic structures than the microtubules in the supporting cells. The microtubule-associated protein MAP-2 is present only in outer hair cells within the organ of Corti and co-localizes with tubulin in these cells. No other MAPs (1,3,4,5) are present. Tau is found in the nerve fibers below both inner and outer hair cells and in the osseous spiral lamina. It is clear that the actin-binding and microtubule-associated proteins present in the cochlea co-localize with actin and tubulin and that they modulate microfilament and microtubule structure and function in a manner similar to that seen in other cell types. The location of some of these proteins in outer hair cells suggests a role for microfilaments and microtubules in outer hair cell motility.

Structural relationships of the unfixed tectorial membrane.

Although the tectorial membrane has a key role in the function of the organ of Corti, its structural relationship within the cochlear partition is still not fully characterised. Being an acellular structure, the tectorial membrane is not readily stained with dyes and is thus difficult to visualise. We present here detailed observations of the unfixed tectorial membrane in an in vitro preparation of the guinea pig cochlea using confocal microscopy. By perfusing the fluid compartments within the cochlear partition with fluorochrome-conjugated dextran solutions, the tectorial membrane stood out against the bright background. The tectorial membrane was seen as a relatively loose structure as indicated by the dextran molecules being able to diffuse within its entire volume. There were, however, regions showing much less staining, demonstrating a heterogeneous organisation of the membrane. Especially Hensen's stripe and regions facing the outer hair cell bundles appeared more condensed. Whereas no connections between Hensen's stripe and the inner hair cell bundles could be observed, there was clearly a contact zone between the stripe and the reticular lamina inside of the inner hair cell.