Stefan Dazert

Basic fibroblast growth factor (FGF‐2) protects rat cochlear hair cells in organotypical culture from aminoglycoside injury

Given the evidence that basic fibroblast growth factor (FGF‐2) can protect neural and retinal cells from degeneration, we evaluated the potential of this growth factor to protect sensory cells in the inner ear. When sensory cells of the organ of Corti are exposed to aminoglycoside antibiotics such as neomycin either in vivo or in vitro, significant ototoxicity is observed. The in vitro cytotoxic effects of neomycin are dose and time dependent. In neonatal rat organ of Corti cultures, complete inner and outer hair cell destruction is observed at high (mM) concentrations of neomycin while inner hair cell survival and severely damaged outer hair cells are noted at moderate (μM) concentrations, with a maximal effect observed after 2 days of culture. Approximately 50% of cochlear outer hair cells are lost at a dose of 35 μM neomycin, and most surviving cells show disorganized stereocilia. Inner hair cells show primarily disorganization of their stereocilia. A significant protective effect is observed when the organ of Corti is pre‐treated with FGF‐2 (500 ng/ml) for 48 hours, and then FGF‐2 is included with neomycin in the culture medium. A greater extent of outer hair cell survival and a significant decrease in stereociliary damage are noted with FGF‐2. However, disorganization of inner hair cell stereocilia is unaffected by FGF‐2. The protective effect of FGF‐2 is specific, since interleukin‐1B, nerve growth factor, tumor necrosis factor, and epidermal growth factor are ineffective, while retinoic acid and transforming growth factor alpha show only a moderate protective effect. These results confirm the potential of molecules like FGF‐2 for preventing cell death due to a variety of causes.

EphA4 provides repulsive signals to developing cochlear ganglion neurites mediated through ephrin-B2 and -B3.

The ephrins and Eph receptors make up two large families of bi‐directional signaling molecules that are known to play a role in the development of the nervous system. Recently, expression of EphA4 in the developing cochlea was shown, with strong expression in cells lining the osseous spiral lamina (OSL) through which afferent dendrites must pass to reach the organ of Corti (OC). It was also demonstrated that ephrin‐B2 and ‐B3, both of which are known to interact with EphA4, are expressed by spiral ganglion (SG) neurons. To investigate the functional role of EphA4 in the development of inner ear neurons, neonatal rat SG explants were cultured for 72 hours on uniformly coated surfaces or near stripes of EphA4/IgG‐Fc‐chimera. Control explants were cultured on or near IgG‐Fc and EphA1/IgG‐Fc‐chimera. To assess the roles of ephrin‐B2 and ‐B3 in EphA4 signaling, SG explants were cultured with or without anti‐ephrin‐B2 and/or ‐B3 blocking antibodies. Growth patterns of SG neurites at the border of EphA4 receptor stripes showed repulsion, characterized by turning, stopping and/or reversal. In the case of IgG‐Fc and EphA1, the neurites grew straight onto the stripes. Treatment with either anti‐ephrin‐B2 or ‐B3 blocking antibodies significantly reduced the repulsive effect of an EphA4 stripe. Moreover, when both antibodies were used together, neurites crossed onto EphA4 stripes with no evidence of repulsion. The results suggest that EphA4 provides repulsive signals to SG neurites in the developing cochlea, and that ephrin‐B2 and ‐B3 together mediate this response. J. Comp. Neurol. 462:90–100, 2003.

FOCAL DELIVERY OF FIBROBLAST GROWTH FACTOR-1 BY TRANSFECTED CELLS INDUCES SPIRAL GANGLION NEURITE TARGETING IN VITRO

Sensory cells in the cochlea of the rat transiently express acidic fibroblast growth factor (FGF‐1) during the developmental period of terminal innervation in the sensory epithelium. To explore the potential role of FGF‐1 in terminal innervation events, the response of cochlear ganglion neurons to FGF‐1 was evaluated in culture. Explants from the spiral ganglion of postnatal day 5 rats were cultured in the presence of exogenous FGF‐1, with or without heparin. FGF‐1 in the culture medium produced a dose‐dependent increase in the number and length of neurites produced by spiral ganglion neurons, a response that was enhanced by heparin. To assess the effects of FGF‐1 produced by a focal, cellular source, additional explants were cocultured with 3T3 cell transfectants that secrete FGF‐1. Neurites that came into contact with FGF‐1 secreting cells branched, formed bouton‐like terminal swellings on the surface of the transfectants, and stopped extending. The results suggest that FGF‐1 may stimulate neurite extension into the sensory epithelium of the cochlea and that focal production of FGF‐1 may contribute to the formation of contacts on sensory cells by developing neurites. J. Cell. Physiol. 177:123–129, 1998.

Ras/MEK but not p38 signaling mediates NT-3-induced neurite extension from spiral ganglion neurons.

Neurotrophin (NT)-3 is expressed in the neuronal target tissue of the developing rat cochlea and has been shown to promote the survival and neurite outgrowth of spiral ganglion (SG) neurons, suggesting a role for this protein during the innervation of the organ of Corti. In other neurons, NT-3 can mediate neuritogenesis and survival via a number of intracellular signal pathways. To date, the intracellular transduction pathways involved in the mediation of NT-3 effects have not been investigated in SG neurons. To determine whether the activities of NT-3 on SG neurons are dependent on the activation of mitogen-activated protein kinase kinases (MEK)/extracellular-signal-regulated kinases (ERK), Ras, and/or p38, SG explants from postnatal-day 4 rats were cultured with NT-3 and increasing concentrations of the MEK inhibitor U0126, the Ras farnesyl-transferase inhibitor (FTI)-277, and the p38 inhibitor SB203580. After fixation and immunocytochemical labeling, neurite growth was evaluated. A dose-dependent decrease of the effects of NT-3 on length and number of processes was observed in the U0126- and FTI-277-treated SG neurons. In contrast, SB203580 had no significant effect on NT-3-mediated stimulation of neurite growth, in terms of either number or length. The results suggest that NT-3 effects on SG neurons are mediated primarily by the Ras/MEK/ERK signaling pathway.

Osteoclast stimulating and differentiating factors in human cholesteatoma.

Objectives To investigate the expression of osteoclast‐activating and differentiating factors and to study the occurrence of osteoclast precursor cells and osteoclasts in acquired human cholesteatoma tissue.

Interaction of spiral ganglion neuron processes with alloplastic materials in vitro

The cochlear implant (CI) involves the introduction of alloplastic materials into the cochlea. While current implants interact with cochlear neurons at a distance, direct interactions between spiral ganglion (SG) neurites and implants could be fostered by appropriate treatment with neurotrophic factors. The interactions of fibroblasts and osteoblasts with alloplastic materials have been well studied in vitro and in vivo. However, interactions of inner ear neurons with such alloplastic materials have yet to be described. To investigate survival and growth behavior of SG neurons on different materials, SG explants from post-natal day 5 rat SG were cultured for 72 h in the presence of neurotrophin-3 (10 ng/ml) on titanium, gold, stainless steel, platinum, silicone and plastic surfaces that had been coated with laminin and poly-L-lysine. Neurite outgrowth was investigated after immunohistological staining for neurofilament, by image analysis to determine neurite extension and directional changes. Neurite morphology and adhesion to the alloplastic material were also evaluated by scanning electron microscopy (SEM). On titanium, SG neurites reached the highest extent of outgrowth, with an average length of 662 microm and a mean of 31 neurites per explant, compared to 568 microm and 21 neurites on gold, 574 microm and 24 neurites on stainless steel, 509 microm and 16 neurites on platinum, 281 microm and 12 neurites on silicone and 483 microm and 31 neurites on plastic. SEM revealed details of adhesion of neurites and interaction with non-neuronal cells. The results of this study indicate that the growth of SG neurons in vitro is strongly influenced by alloplastic materials, with titanium exhibiting the highest degree of biocompatibility with respect to neurite extension. The knowledge of neurite interaction with different alloplastic materials is of clinical interest, as development in CI technology leads to closer contact of implanted electrodes with surviving inner ear neurons.

TRANSFECTION OF NEONATAL RAT COCHLEAR CELLS IN VITRO WITH AN ADENOVIRUS VECTOR

A recombinant adenovirus vector containing a βI‐galactosidase reporter gene was used to transfect neonatal rat organ of Corti or spiral ganglion explants in vitro. Infection at appropriate titers (106−107 pfu/ml) transduced virtually all cells in the cultures after 72 hr. However, spiral ganglion neurons and cells in the inner hair cell regions of the organ of Corti showed the highest levels of expression. Viral titers that produced high levels of β‐galactosidase expression did not appear to damage the cultures, and did not inhibit neurite outgrowth from spiral ganglion cells. However, higher titers (108‐109pfu/ml) clearly diminished explant viability and inhibited neurite extension. The results demonstrate that cochlear cells can be transfected successfully with an adenovirus vector, at viral titers which do not induce obvious signs of cellular damage or dysfunction.

Cochlear spiral ganglion cell degeneration in wild-caught mice as a function of age.

Presbyacusis in humans is an age-related bilateral sensorineural hearing impairment generally associated with degeneration of cochlear hair cells and spiral ganglion cells (SGC) predominantly in the basal turn but present in the apical turn. Investigations of cochleas of aged rats and gerbils reveal a large loss of SGCs in the apical as well as the basal turns. Genetically inbred aged mice, on the other hand, seem to have variable amounts of SGC loss beginning in some strains very early in the life span of the animals and greatest in the basal turn. Three age groups of wild-caught, then laboratory-bred, mice were investigated to determine the pattern of SGC degeneration. In 18-19-month-old animals the main loss of SGCs occurred in the basal turn (49% loss compared to 2-3 months) followed by the apical turn (31%). The greatest SGC losses in the 28-31-month-old animals were in both the apical (76%) and basal turns (74%). Thus, this strain of mice is similar to other rodents in that both ends of the ganglion are affected by SGC degeneration associated with aging.

Astringency Is a Trigeminal Sensation That Involves the Activation of G Protein–Coupled Signaling by Phenolic Compounds

Astringency is an everyday sensory experience best described as a dry mouthfeel typically elicited by phenol-rich alimentary products like tea and wine. The neural correlates and cellular mechanisms of astringency perception are still not well understood. We explored taste and astringency perception in human subjects to study the contribution of the taste as well as of the trigeminal sensory system to astringency perception. Subjects with either a lesion or lidocaine anesthesia of the Chorda tympani taste nerve showed no impairment of astringency perception. Only anesthesia of both the lingual taste and trigeminal innervation by inferior alveolar nerve block led to a loss of astringency perception. In an in vitro model of trigeminal ganglion neurons of mice, we studied the cellular mechanisms of astringency perception. Primary mouse trigeminal ganglion neurons showed robust responses to 8 out of 19 monomeric phenolic astringent compounds and 8 polymeric red wine polyphenols in Ca(2+) imaging experiments. The activating substances shared one or several galloyl moieties, whereas substances lacking the moiety did not or only weakly stimulate responses. The responses depended on Ca(2+) influx and voltage-gated Ca(2+) channels, but not on transient receptor potential channels. Responses to the phenolic compound epigallocatechin gallate as well as to a polymeric red wine polyphenol were inhibited by the Gαs inactivator suramin, the adenylate cyclase inhibitor SQ, and the cyclic nucleotide-gated channel inhibitor l-cis-diltiazem and displayed sensitivity to blockers of Ca(2+)-activated Cl(-) channels.

In vivo adenoviral transduction of the neonatal rat cochlea and middle ear

Virally mediated gene transfer to the adult mammalian ear appears to be a powerful strategy to investigate gene function in the auditory system and to develop new therapeutic treatment for hearing impaired patients. However, there has been little work done in the neonatal middle and inner ear. In this study, a recombinant adenoviral (AdV) vector was used for gene transfer of a beta-galactosidase (beta-gal) reporter gene to the neonatal middle ear and cochlea of 5 day old rats. For transduction of middle ear, AdV was injected through the tympanic membrane into the tympanic cavity. Three and 7 days later, strong expression of beta-gal was observed in epithelial cells of the mucosa, but not in the underlying stroma or mesenchyme. There was little or no infiltration of leukocytes. No expression of beta-gal was detected inside the cochlea or vestibular system. When AdV was injected into the basal turn of the cochlea, high levels of beta-gal expression were observed in cells lining the perilymphatic space and in parts of the spiral ligament 3, 7 and 21 days later. Spiral ganglion cells did not express beta-gal. However, virally mediated gene transfer was observed in some cells of the organ of Corti. A moderate infiltration of leukocytes into the labyrinth was observed, but no vestibular or auditory dysfunction. These results demonstrate that neonatal middle ear and cochlear cells can be successfully transduced with an AdV vector in vivo, without obvious morphological signs of inflammation or cellular damage. AdV vectors provide a tool for investigation of the role of genes in influencing the development of middle and inner ear structures. Virally mediated expression of protective genes could also be used to rescue hair cells or spiral ganglion cells from congenital degeneration or damage.