Lynne M. Bianchi

EphB2 guides axons at the midline and is necessary for normal vestibular function.

Mice lacking the EphB2 receptor tyrosine kinase display a cell-autonomous, strain-specific circling behavior that is associated with vestibular phenotypes. In mutant embryos, the contralateral inner ear efferent growth cones exhibit inappropriate pathway selection at the midline, while in mutant adults, the endolymph-filled lumen of the semicircular canals is severely reduced. EphB2 is expressed in the endolymph-producing dark cells in the inner ear epithelium, and these cells show ultrastructural defects in the mutants. A molecular link to fluid regulation is provided by demonstrating that PDZ domain-containing proteins that bind the C termini of EphB2 and B-ephrins can also recognize the cytoplasmic tails of anion exchangers and aquaporins. This suggests EphB2 may regulate ionic homeostasis and endolymph fluid production through macromolecular associations with membrane channels that transport chloride, bicarbonate, and water.

Comparison of Ephrin-A ligand and EphA receptor distribution in the developing inner ear

Members of the recently discovered Eph family appear to play important roles in a variety of developmental processes including tissue segmentation, cell migration and axonal guidance. To begin to understand the functions of the EphA subclass of receptors and their corresponding GPI‐linked (ephrin‐A) ligands in the inner ear, a developmental immunohistochemical analysis was completed. The results indicated that the ligands ephrin‐A1 and ephrin‐A2 were localized mainly at cellular boundaries in the inner ear. Ephrin‐A1 was detected mainly in the epithelial cells lining the fluid filled ducts of the inner ear, whereas ephrin‐A2 was prominently expressed in connective tissue regions. The receptor EphA4 was detected in vestibular hair cells. EphA5 and EphA7 were detected mainly in cochlear and vestibular supporting cells. These results suggest that these Eph molecules play a role in establishing the formation and cellular organization of the complex inner ear labyrinth. Additionally, all of the ligands and receptors evaluated were expressed in vestibular and cochlear neurons at various developmental stages, suggesting they may play a role in establishing or maintaining innervation to the inner ear. Anat Rec 254:127–134, 1999.

Microwave decalcification of human temporal bones.

Objectives/Hypothesis Morphological and immunohistochemical studies of human temporal bones are challenging as a result of difficulties in obtaining reliably fi‐ed specimens and the lengthy time required for decalcification, typically 4 to 7 months. A novel method of processing human temporal bones using a microwave oven to accelerate decalcification is described. This procedure provides a rapid means of decalcifying temporal bones with good preservation of tissue morphology and antigenicity.

Methods for providing therapeutic agents to treat damaged spiral ganglion neurons.

Sensorineural hearing loss, characterized by damage to sensory hair cells and/or associated nerve fibers is a leading cause of hearing disorders throughout the world. To date, treatment options are limited and there is no cure for damaged inner ear cells. Because the inner ear is a tiny organ housed in bone deep within the skull, access to the inner ear is limited, making delivery of therapeutic agents difficult. In recent years scientists have investigated a number of growth factors that have the potential to regulate survival or recovery of auditory neurons. Coinciding with the focus on molecules that may restore function are efforts to develop novel delivery methods. Researchers have been investigating the use of mini osmotic pumps, viral vectors and stem cells as a means of providing direct application of growth factors to the inner ear. This review summarizes recent findings regarding the molecules that may be useful for restoring damaged spiral ganglion neurons, as well as the advantages and disadvantages of various delivery systems.

Embryonic Inner Ear Cells Reaggregate into Specific Patterns In Vitro

The sensory epithelia of the mammalian inner ear consist of a highly precise pattern of sensory hair cells and supporting cells. The mechanisms regulating this patterning are only beginning to be determined. The present study describes a method for culturing dissociated embryonic inner ear cells and the resulting patterning that occurs in these cultures. The results indicate that developing inner ear cells aggregate into precise patterns on a two-dimensional substrate, suggesting that intrinsic patterning mechanisms remain active in vitro. Using antibodies and scanning electron microscopy to detect hair cells and nonsensory cells, it was determined that only a subset of aggregates contained sensory hair cells. The hair cells were organized into specific patterns and surrounded by supporting cells, similar to the in vivo pattern. Additionally, hair cells increased their immunoreactivity and number of stereocilia over time, suggesting that hair cells continue to mature in vitro. Thus, the study reveals that the cells of the developing inner ear provide the necessary signals that direct sensory hair cells and supporting cells to reassociate into very precise patterns in vitro and that these patterns are reminiscent of the patterning that occurs in vivo.

Analysis of BDNF production in the aging gerbil cochlea.

Degeneration of cochlear neurons is the most commonly observed cellular change in the aging human and gerbil cochlea. Although it is unclear what leads to this neuronal loss, changes in the production of target-derived trophic factors may be the ultimate cause of cochlear neuron degeneration. The present study used an enzyme-linked immunosorbent assay to investigate whether BDNF is produced by the organ of Corti or cochlear ganglia of young, middle aged, or aged gerbils. The results revealed an age-related increase in BDNF in the organ of Corti, but not in the cochlear ganglia.

Selective and transient expression of a native chondroitin sulfate epitope in Deiters' cells, pillar cells, and the developing tectorial membrane

The tectorial membrane (TM) is an acellular connective tissue overlying the sensory hair cells of the organ of Corti. Association of the tectorial membrane with the stereocilia of the sensory hair cells is necessary for proper auditory function. During development, the mature tectorial membrane is thought to arise by fusion of a “major” and “minor” tectorial membrane (Lim, Hear Res 1986;22:117–146). Several proteins and glycoconjugates have been detected in the developing TM; however, the specific molecules which mediate fusion of the two components of the TM have not been identified.

Embryonic inner ear cells use migratory mechanisms to establish cell patterns in vitro.

The hair cells of the sensory epithelium in the inner ear are among the most precisely organized cells in vertebrates. The mechanisms that lead to this orderly arrangement are only beginning to be understood. It has been suggested that hair cells use migratory mechanisms to help achieve their final position in the organ of Corti. The small size and complex organization of the intact inner ear have made it difficult to monitor changes in hair cell location over time in vivo. In the present study, an established in vitro assay of dissociated, embryonic inner ear cells was used to monitor how hair cells reorganize over time. The hair cell specific marker myosin‐VI demonstrated that hair cell precursors from both cochlear and vestibular regions reorganized into specific patterns between 3–24 hr in vitro. In contrast to the unlabeled cells, the myosin‐VI‐positive cells extended processes while establishing the hair cell patterning within an aggregate. These studies support the hypothesis that hair cell precursors actively migrate to help achieve final patterning within the inner ear sensory epithelium.

R440 – Generation of Neuron-Like Cells in Spiral Ganglion Cultures

Problem Development of the auditory nerve is dependent on neurotrophic factors. Neurotrophins BDNF and NT-3 are critical in the later stages of development. More recently, a substance secreted by the early inner ear, otocyst-derived factor (ODF), was shown to stimulate development of primitive auditory neurons at the earliest stages. We hypothesized that this powerful neurotrophic substance might be capable of regenerating auditory neurons in the mature animal. Methods Cultured neurons and whole explants from neonatal mouse spiral ganglia were incubated with either BDNF or supernatant from an ODF-secreting cell line. Results Exposure to ODF resulted in large numbers of cells which stained with neuronal markers, and had neuronal morphology. Though they appeared somewhat different from the native spiral ganglion neurons seen in BDNF-treated cultures, they were present in vastly greater numbers, and appeared to arise from within the proliferating, migrating glial cell populations growing along with the neurons. These cells were not seen in cultures containing either control serum or BDNF. Addition of beta-bungarotoxin, a neurotoxin, to spiral ganglia just after harvest destroyed the native neurons, which did not regenerate upon addition of BDNF. However, many of the new neuron-like cells were observed after rescue with ODF, suggesting they represented a newly regenerated population of cells. Conclusion These data suggest that the components of ODF have the potential to regenerate neuronal cells, possibly from precursors or stem cells existing within the supporting cell populations of the auditory nerve. Significance The ability to regenerate auditory neurons would have exciting implications on the design and function of cochlear implants. Work continues in our lab to better define the properties of these new cells, and to isolate ODFs active component growth factors. Support Commonwealth of Pennsylvanias Tobacco Formula Fund.