Biomedical subjects

M Ulfendahl

Publications and source records attributed to M Ulfendahl.

At least 19 recordsLinked to original sources

PubMed2006

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.

Acoustic Stimulation↗

PubMed2006

Lateral diffusion anisotropy and membrane lipid/skeleton interaction in outer hair cells.

The organization of the plasma membrane of cells in lipid domains affects the way the membrane interacts with the underlying protein skeleton, which in turn affects the lateral mobility of lipid and protein molecules in the membrane. Membrane fluidity properties can be monitored by various approaches, the most versatile of which is fluorescence recovery after photobleaching (FRAP). We extended previous FRAP experiments on isolated cochlear outer hair cells (OHCs) by analyzing the two-dimensional pattern of lipid diffusion in the lateral membrane of these cells. We found that membrane lipid mobility in freshly isolated OHCs is orthotropic, diffusion being faster in the axial direction of the cell and slower in the circumferential direction. Increasing the cell's turgor pressure by osmotic challenge reduced the axial diffusion constant, but had only a slight effect on circumferential diffusion. Our results suggest that lipid mobility in the OHC plasma membrane is affected by the presence of the cell's orthotropic membrane skeleton. This effect could reflect interaction with spectrin filaments or with other membrane skeletal proteins. We also performed a number of FRAP measurements in temporal bone preparations preserving the structural integrity of the hearing organ. The diffusion rates measured for OHCs in this preparation were in good agreement with those obtained in isolated OHCs, and comparable to the mobility rates measured on the sensory inner hair cells. These observations support the idea that the plasma membranes of both types of hair cells share similar highly fluid phases in the intact organ. Lipid mobility was significantly slower in the membranes of supporting cells of the organ of Corti, which could reflect differences in lipid phase or stronger hindrance by the cytoskeleton in these membranes.

Animals↗

PubMed2006

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.

Algorithms↗

PubMed2002

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.

Adaptation, Physiological↗

PubMed2001

Image restoration for confocal microscopy: improving the limits of deconvolution, with application to the visualization of the mammalian hearing organ.

Deconvolution algorithms have proven very effective in conventional (wide-field) fluorescence microscopy. Their application to confocal microscopy is hampered, in biological experiments, by the presence of important levels of noise in the images and by the lack of a precise knowledge of the point spread function (PSF) of the system. We investigate the application of wavelet-based processing tools to deal with these problems, in particular wavelet denoising methods, which turn out to be very effective in application to three-dimensional confocal images. When used in combination with more classical deconvolution algorithms, these methods provide a robust and efficient restoration scheme allowing one to deal with difficult imaging conditions. To make our approach applicable in practical situations, we measured the PSF of a Biorad-MRC1024 confocal microscope under a large set of imaging conditions, including in situ acquisitions. As a specific biological application, we present several examples of restorations of three-dimensional confocal images acquired inside an intact preparation of the hearing organ. We also provide a quantitative assessment of the gain in quality achieved by wavelet-aided restorations over classical deconvolution schemes, based on a set of numerical experiments that we performed with test images.

Algorithms↗

PubMed2001

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.

Animals↗

PubMed2000

[Future cure of hearing disorders? Gene therapy and stem cell implantation are possible new therapeutic alternatives].

Hearing loss is a very common disorder; nearly 10 per cent of the population is affected. Recently, a few findings such as the roles of neurotrophins, nitric oxide, reactive oxygen species and glutamate receptors in the peripheral hearing system have been highlighted. In this review, focus is set on possible mechanisms of peripheral hearing disorders, and on recent advances to prevent and treat hearing loss. Clinically useful treatment strategies, especially gene therapy and the use of embryonic stem cells, are particularly stressed.

Animals↗

PubMed2000

Perilymphatic fluid compartments and intercellular spaces of the inner ear and the organ of Corti.

An in vitro preparation of the inner ear cochlea has been used to visualize the structural relationships of unfixed, living sensory cells and structural components within the intact hearing organ. By perfusing perilymphatic compartments of the cochlea with fluorochrome-conjugated dextran, the extracellular spaces were clearly outlined. The staining pattern illustrated the large fluid compartments formed by the tunnel of Corti, the space of Nuel, and the outer tunnel. The dextran solution also indicated the spaces between the outer hair cell rows, the inner hair cells, and the surrounding supporting cells. The staining pattern demonstrates that the organ of Corti has a loose structure, suggesting a weak mechanical coupling between the cells. Moreover, it is evident that substances applied to the perilymph (e.g., therapeutic drugs) will readily reach all the cells of the hearing organ. In addition to the intraorgan fluid compartments, the spiral limbus was shown to contain significant volumes of perilymph within the intercellular spaces forming the so-called teeth of Huschke between the interdental cells. An extensive system of bundles following the teeth of Huschke was shown to be completely immersed in perilymph. The bundles were stained by a potentiometric dye, which in the inner ear primarily stains nerve fibers and sensory cells, which may indicate a nervous control of cells in this region.

Animals↗

PubMed2000

An in vitro preparation to access cellular and neuronal components in the mouse inner ear.

An in vitro mouse temporal bone preparation has been developed in order to allow the investigation of structures and functions in a living hearing organ. Fluorescent vital probes (a potentiometric styryl dye, RH 795, and a vital dye staining cellular cytoplasm, calcein) were perfused through the scala tympani to stain cellular components of the cochlea. Observations of the cochlear apical turn were performed using a confocal microscope. In spite of the anatomical constraints due to the small size of the mouse cochlea, detailed images were obtained. The sensory cells as well as their innervating nerve fibres were clearly seen. Nerve fibres crossing the tunnel of Corti and projecting to the outer hair cells could also be visualised.

Animals↗

PubMed1999

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.

Acoustic Stimulation↗

PubMed1999

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.

Animals↗

PubMed1998

Acoustic overstimulation increases outer hair cell Ca2+ concentrations and causes dynamic contractions of the hearing organ.

The dynamic responses of the hearing organ to acoustic overstimulation were investigated using the guinea pig isolated temporal bone preparation. The organ was loaded with the fluorescent Ca2+ indicator Fluo-3, and the cochlear electric responses to low-level tones were recorded through a microelectrode in the scala media. After overstimulation, the amplitude of the cochlear potentials decreased significantly. In some cases, rapid recovery was seen with the potentials returning to their initial amplitude. In 12 of 14 cases in which overstimulation gave a decrease in the cochlear responses, significant elevations of the cytoplasmic [Ca2+] in the outer hair cells were seen. [Ca2+] increases appeared immediately after terminating the overstimulation, with partial recovery taking place in the ensuing 30 min in some preparations. Such [Ca2+] changes were not seen in preparations that were stimulated at levels that did not cause an amplitude change in the cochlear potentials. The overstimulation also gave rise to a contraction, evident as a decrease of the width of the organ of Corti. The average contraction in 10 preparations was 9 microm (SE 2 microm). Partial or complete recovery was seen within 30-45 min after the overstimulation. The [Ca2+] changes and the contraction are likely to produce major functional alterations and consequently are suggested to be a factor contributing strongly to the loss of function seen after exposure to loud sounds.

Acoustic Stimulation↗

PubMed1998

Axial and transverse stiffness measures of cochlear outer hair cells suggest a common mechanical basis.

The function of the hearing organ is based on mechanical processes occurring at the cellular level. The mechanical properties of guinea-pig isolated sensory cells were investigated using two different techniques. The stiffness of the outer hair cells along the longitudinal axis was measured by compressing the cell body using stiffness-calibrated quartz fibres. For cells with a mean length of 69 micron, the mean axial compression stiffness was 1. 1+/-0.8 mN/m (+/-SD). There was an inverse relation between stiffness and cell length. The stiffness of the cell membrane perpendicular to the longitudinal axis of the sensory cell was measured by indenting the cell membrane with a known force. The mean lateral indentation stiffness was 3.3+/-1.5 mN/m (+/-SD) for cells with a mean length of 64 microm. Longer cells were less stiff than short cells. Modelling the hair cell as a shell with bending resistance, finite element calculations demonstrated that the axial compression stiffness correlated well with the lateral indentation stiffness, and that a simple isotropic model is sufficient to explain the experimental observations despite the different stress strain states produced by the two techniques. The results imply that the two different stiffness properties may originate from the same cytoskeletal structures. It is suggested that the mechanical properties of the outer hair cells are designed to influence the sound-induced motion of the reticular lamina. In such a system, stiffness changes of the outer hair cell bodies could actively control the efficiency of the mechanical coupling between the basilar membrane and the important mechanoelectrical transduction sites at the surface of the hearing organ.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

Vibration of reflective beads placed on the basilar membrane.

Most investigators place reflective beads on the basilar membrane to measure its vibration with optical methods. It is therefore important to find out if the beads faithfully follow the motion of the structures on which they are placed. Vibration of the beads on the basilar membrane and basilar membrane adjacent to the beads are measured in the third turn of the guinea pig cochlea in a temporal bone preparation. It is shown that the beads do not follow the motion of the organ. The mechanism by which this departure may occur is investigated by modeling the motion of the beads on the Claudius' cells.

Acoustic Stimulation↗

PubMed1998

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.

Animals↗

PubMed1998

An in vitro model for acoustic overstimulation.

Although many studies have been performed on the effects of acoustic overstimulation on the inner ear, our knowledge about the cellular processes underlying reduced hearing sensitivity and auditory cell death is still limited. In order to further our understanding of cellular processes occurring in conjunction with acoustic trauma, we designed an in vitro model to study the effects of overstimulation directly on sensory hair cells isolated from the low-frequency part of the guinea pig cochlea. The isolated outer hair cells were subjected to pressure jets delivered by a glass micropipette positioned close to the cell, in order to mimic the pressure changes occurring in the intact inner ear during sound stimulation. A second micropipette coupled to a piezoresistive pressure transducer was used as a probe measuring the pressure at precise locations at and around the cell. In a previous study, we found that such stimulation gave rise to increases in the intracellular calcium concentration. The present study characterizes the stimulus, describes the computer-controlled setup used for calibration, and gives examples of different modes of overstimulation at the cellular level. The peak pressure that could be generated using the pressure jet was around 325 Pa, or 144 dB (re 20 microPa) at 140 Hz. The pressure jet elicited large mechanical vibrations of the cell bodies of isolated cells. The vibration mode of the cells often changed over time, implying that the stimulation caused changes of the cellular stiffness. However, most cells appeared quite resistant to the high intensity mechanical stimulation.

Acoustic Stimulation↗