Direct round window application of gentamicin with varying delivery vehicles: a comparison of ototoxicity.

Objective: To evaluate patterns of ototoxic damage sustained from gentamicin applied directly to the round window in varying delivery vehicles.

Setting: The search for a reliable method of drug delivery to the inner ear continues. Vehicles such as Gelfoam, hyaluronic acid, and fibrin may allow for a more reproducible pattern of ototoxic damage.

Microtubule cold stability in supporting cells of the gerbil auditory sensory epithelium: correlation with tubulin post-translational modifications.

Sensory cells in the organ of Corti exhibit loose microtubule networks enriched in tyrosinated tubulin, whereas supporting cells have bundled microtubules containing post-translationally modified tubulin. The tubulin isoform distribution suggests that the microtubules in sensory cells are dynamic and those in supporting cells are stable. To test this, microtubule resistance to cold-induced depolymerization was examined by using immunocytochemical methods and antibodies to post-translationally modified tubulins.

Auditory nerve fibers in young and quiet-aged gerbils: morphometric correlations with endocochlear potential.

The number, size and distribution of myelinated nerve fibers were analyzed in the osseous spiral lamina (OSL) of young and old gerbils raised in a quiet environment. Because decreased endocochlear potentials (EPs) play a significant role in age-related hearing loss in the gerbil, we correlated morphometric and topographical data for nerve fibers with EP measurements in the same ear. Fibers were analyzed at the 2 and 10 kHz locations.

Anatomic and morphometric changes to gerbil posterior cristas following transtympanic administration of gentamicin and streptomycin.

Many studies have sought to document ototoxic damage and to study repair and regeneration of mammalian vestibular sensory epithelia. However, linear density analysis of the sensory cells or use of methods that focus on detection of actin in the stereocilia and cuticular plates at the reticular lamina detect only the disappearance of "hair cells" as defined by a narrow set of criteria. The research presented here focuses on the effects of two ototoxic drugs (gentamicin and streptomycin).

Immunocytochemical identification of proteins within the Pacinian corpuscle.

Light- and electron-microscopic immunocytochemistry (ICC) was performed on Pacinian corpuscles (PCs) obtained from cat mesentery to determine the presence and location of various proteins within the accessory capsule and the neurite. Antibodies to tubulin, neurofilament 200, actin, collagen II and V, glial fibrillary acidic protein (GFAP) and S-100 were used. Type II collagen was localized only in the outer core of the accessory capsule, which is composed of an inner core, an intermediate layer or growth zone, an outer core and an external capsule.

Study of afferent nerve terminals and fibers in the gerbil cochlea: distribution by size.

The purpose of the present study was to determine if the synaptic terminals and nerve fibers in the gerbil cochlea fall into morphologically and spatially classified groups. In cats and guinea pigs, these groups, based on size, location on inner hair cell (IHC) and stratification within the osseous spiral lamina, have been found to correlate with spontaneous rate, threshold sensitivity and projection pattern to the cochlear nucleus. Thus, there may be anatomical data to suggest mechanisms for intensity coding of different frequencies of sound.

Age-related changes in microtubules in the guinea pig organ of Corti. Tubulin isoform shifts with increasing age suggest changes in micromechanical properties of the sensory epithelium.

Biochemical and immunocytochemical analyses have been used to provide new insights into age-related changes in the sensory and supporting cells of the guinea pig organ of Corti. Quantitative densitometry of immunoblots showed that, while levels of alpha-tubulin remained relatively constant in guinea pigs from 3 weeks to 18 months old, there were progressive shifts in some tubulin isoforms. Levels of tyrosinated tubulin increased with age, nontyrosinatable tubulin (delta2-tubulin) showed a compensatory decrease, but detyrosinated tubulin did not change; acetylated, polyglutamylated, and glycylated tubulin levels also decreased.

Histologic changes after semicircular canal occlusion in guinea pigs.

Hypothesis: Histologic changes occurring after varying degrees of surgical trauma to the inner ear in guinea pigs can reveal the mechanism of hearing preservation/loss.

Background: Surgical approaches to the inner ear that allow for hearing preservation have gained increasing acceptance in neurotologic surgery. The mechanisms responsible for hearing preservation and hearing loss after partial labyrinthectomy are as yet poorly understood.

Olivocochlear neurons in the chinchilla: a retrograde fluorescent labelling study.

Although the chinchilla is widely used as a model for auditory research, little is known about the distribution and morphology of its olivocochlear neurons. Here, we report on the olivocochlear neurons projecting to one cochlea, as determined by single and double retrograde fluorescent tracer techniques. 10 adult chinchillas were anesthetized and given either unilateral or bilateral injections of a fluorescent tracer (either Fluoro-Gold or Fast Blue) into scala tympani or as a control, a unilateral injection into the middle ear cavity.

Comparison of vestibular and cochlear ototoxicity from transtympanic streptomycin administration.

Hypothesis: The relative dose-related cochlear and vestibular ototoxicity produced by transtympanically injected streptomycin (SM) compared to that of gentamicin (GM) was assessed.

Background: Although SM, the first aminoglycoside used transtympanically, is thought to be selectively vestibulotoxic, it has been replaced by GM in current clinical use. Little experimental data exist that directly demonstrate the relative cochlear and vestibular ototoxicity resulting from transtympanic administration of SM compared to GM.

Study of the gerbil utricular macula following treatment with gentamicin, by use of bromodeoxyuridine and calmodulin immunohistochemical labelling.

Effects of ototoxic drugs on the gerbil vestibular sensory epithelium were probed by use of immunocytochemical labelling with antibodies to both a mitogenic marker (bromodeoxyuridine) and a hair cell specific protein (calmodulin). Nine animals had gentamicin administered once daily for 5 days, as a transtympanic injection into the right middle ear. They additionally were given a daily intraperitoneal injection of bromodeoxyuridine, starting on the same day as the gentamicin injection and continuing until the day of sacrifice.

Immunocytochemical localization of calmodulin in the vestibular end-organs of the gerbil.

This study demonstrates the presence of calmodulin in the vestibular end-organs of the gerbil by use of immunocytochemistry. Using fluorescence microscopy, calmodulin was localized to the cytoplasm, cuticular plate, and stereocilia of both type I and type II hair cells in the sensory epithelia of the utricle and cristae ampullaris; no label was found in the supporting cells, the dark cells, or the nerve fibers. There was no immunoreactive distinction between the labeling of type I and type II hair cells in the striolar or extrastriolar regions.

Dose-related vestibular and cochlear effects of transtympanic gentamicin.

Hypothesis: To test the relative dose-related cochlear and vestibular ototoxicity produced by transtympanically injected gentamicin in the Mongolian gerbil.

Background: Transtympanic gentamicin is gaining favor as a relatively noninvasive treatment for Meniere's disease (MD). Few basic science studies exist regarding the vestibular and cochlear toxicities and dosage and administration schedules, however.

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines.

Localization of microtubules containing posttranslationally modified tubulin in cochlear epithelial cells during development.

In the adult gerbil inner ear, hair cell microtubules contain predominantly tyrosinated tubulin while supporting cell microtubules contain almost exclusively other isoforms. This cell-type specific segregation of tubulin isoforms is unusual, and in this respect the sensory and supporting cells in this sensory organ differ from other cells observed both in vivo and in vitro. Thus, we hypothesized there must be a shift in the presence and location of tubulin isoforms during development, directly associated with the onset of specialized functions of the cells.

[Immunohistochemical labeling of inner ear tissues embedded in polyethylene glycol 4000--comparative study with araldite and unicryl embedded sections].

An improved method for embedding specimens in polyethylene glycol (PEG) 4000, a water soluble polymer, was used to prepare the vestibular end-organs of the inner ear. Staining of the tissue sections of PEG embedded specimens with antibodies to alpha-tubulin and to calmodulin was compared with staining of tissue sections of Araldite and Unicryl embedded specimens. PEG embedded sections revealed sensitive immunocytochemical labeling with excellent morphological resolution.

Post-translational modifications of tubulin suggest that dynamic microtubules are present in sensory cells and stable microtubules are present in supporting cells of the mammalian cochlea.

Post-translational modifications to tubulin in the sensory and supporting cells of the cochlea were studied using antibodies specific to the tyrosinated, detyrosinated, acetylated and polyglutamylated isoforms. In the sensory cells, microtubules which label intensely with antibodies to tyrosinated tubulin are found in networks within the cytoplasm. Microtubules which label with antibodies to detyrosinated tubulin and polyglutamylated tubulin, but not acetylated tubulin, form a small component of the microtubules found in the cytoplasm only in the region below the cuticular plate.

Cytoskeletal and calcium-binding proteins in the mammalian organ of Corti: cell type-specific proteins displaying longitudinal and radial gradients.

Whole mounts and tissue sections of the organ of Corti from two representative mammalian species, the Mongolian gerbil (Meriones unguiculatus) and the guinea pig (Cavea porcellus) were probed with antibodies to cytoskeletal and calcium-binding proteins (actin, tubulin, including post-translational modifications, spectrin, fimbrin, calmodulin, parvalbumin, calbindin, S-100 and calretinin). All of the proteins tested were expressed in both species. New findings include the following.

Immunocytochemical comparison of posttranslationally modified forms of tubulin in the vestibular end-organs of the gerbil: tyrosinated, acetylated and polyglutamylated tubulin.

Specific antibodies against alpha-tubulin, acetylated alpha-tubulin, tyrosinated alpha-tubulin and polyglutamylated alpha- and beta-tubulin were used to compare the distribution of posttranslationally modified tubulin in the vestibular end-organs of the gerbil. Antibodies to acetylated tubulin labeled a dense network of microtubules in the hair cells and bundles of microtubule in the supporting cells. Nerve fibers within and below the epithelium were weakly labeled.

A simplified method for obtaining 0.5-microns sections of small tissue specimens embedded in PEG.

We describe a new method for embedding small specimens in polyethylene glycol (PEG) 4000. This method preserves cell morphology and provides sensitive immunocytochemical labeling with excellent subcellular resolution. Small tissues are embedded in agarose so that they can be grouped together and oriented for sectioning before infiltration with PEG 4000, a water-soluble polymer.

Three microtubule-organizing centres collaborate in a mouse cochlear epithelial cell during supracellularly coordinated control of microtubule positioning.

Large cell surface-associated microtubule bundles that include about 3,000 microtubules assemble in certain epithelial cells called inner pillar cells in the mouse organ of Corti. Microtubule-organizing centres (MTOCs) at both ends and near the middle of each cell act in concert during control of microtubule positioning. In addition, the three cell surface-associated microtubule-organizing centres are involved in coordinating the connection of bundle microtubules to cytoskeletal components in neighbouring cells and to a basement membrane.

Expression of actin isoforms in the guinea pig organ of Corti: muscle isoforms are not detected.

The specificity of antibodies to actin was assayed by use of immunoblots and histological sections of control tissues enriched for each of six different isoforms. On immunoblots, all antibodies stained at most one band of protein in most of the control materials, with a molecular weight of approximately 43 kDa. Their pattern of staining of muscle and nonmuscle tissues indicated their isoform specificity.

Semi-serial electron-micrographic reconstruction of putative transducer sites in Pacinian corpuscles.

The Pacinian corpuscle (PC) is composed of an afferent neurite surrounded by an accessory capsule formed by concentric layers of lamellae. Projecting from the neurite, which is elliptical in cross-section, are "filopodia" or axonal, spike-like extensions. These filopodia are the putative sites of transduction.

Evidence for calcium-binding proteins and calcium-dependent regulatory proteins in sensory cells of the organ of Corti.

Calcium is thought to play a major signaling role in outer hair cells to control metabolism, cytoskeletal integrity, cell shape and cell excitability. For this to happen, in resting cells the concentration of free calcium ions must be maintained at low levels so that focal increases can trigger specific events. In this paper, the localization of calcium, calcium-binding and calcium-dependent regulatory proteins in sensory cells from the guinea pig inner ear was demonstrated using immunocytochemical and histochemical techniques.