Effect of violation of the labyrinth on the sensory epithelium in the chick cochlea.

Models in which a single large systemic dose of gentamicin is used to cause near-synchronous hair cell (HC) loss in the basal end of the chick cochlea have proven increasingly useful in the study of HC regeneration. We quantified the amount of HC death, as a percentage of the length of the basilar papilla, following single doses of 200 mg/kg and 300 mg/kg of gentamicin in 23-day-old chicks. Following 200 mg/kg of gentamicin, there was total HC loss in the basal 18.0% of the sensory epithelium and partial HC loss in the basal 26.3%. Following 300 mg/kg of gentamicin, there was total HC loss in the basal 30.5% of the epithelium and partial HC loss in the basal 40.9%. The second goal of this study was to determine whether cannula implantation in the inner ear, and infusion of bromodeoxyuridine causes HC damage. We found that creation of a fistula in the labyrinth is not associated with HC damage, but that cannula implantation can cause HC death, and can also cause potentiation of gentamicin-induced HC death. Revision of the cannula and surgical technique to ensure minimal penetration into the labyrinth almost entirely eliminated these effects. We conclude that surgical technique is critical in experimental models in which the labyrinth is violated.

Endotracheal isoflurane anesthesia for chick auditory surgery.

Survival surgeries upon chicks are commonly used in auditory research. Appropriate anesthesia is usually obtained with intramuscular or intraperitoneal injections of systemic agents. These techniques have several drawbacks, including delayed onset of anesthesia, difficulty in adjusting the dosage to accomodate individual animals' different responses, prolonged recovery times, and in some cases substantial mortality. We present a technique of administering inhaled isoflurane via an endotracheal tube which we have used for over a year with excellent results. With this agent, onset of deep anesthesia is very rapid, dosage can be titrated readily, overdosage is survivable, complete recovery occurs within a few minutes and mortality is rare. This technique may be valuable for other auditory scientists performing survival surgery in avian species.

Regulation of p27Kip1 during gentamicin mediated hair cell death.

The INK4 and Kip/Cip families of Cyclin Dependent Kinase inhibitors (CKIs) are regulators of the cell cycle. In addition, CKIS including p27(Kip1) can protect cells from apoptosis in vitro. However, little is known about protective effect of p27(Kip1) in vivo. We used systemic treatment with aminoglycosides to induce hair-cell death in the basilar papilla (BP), the auditory organ of the avian inner ear, and characterised the expression of p27(Kip1) with confocal and immunofluorescence microscopy. In contrast to the adult mammalian cochlea where p27(Kip1) is expressed only in supporting cells, p27(Kip1) is found in the nuclei of both hair cells and supporting cells in the BP of the normal, mature bird. Forty-eight hours after gentamicin treatment, hair cells with TUNEL positive nuclei and hair cells with pyknotic nuclei were both detected, suggesting many hair cells die by apoptosis. When the BP was double labelled for p27(Kip1) and myosin VIIa, a hair-cell specific protein, all dying hair cells that had been ejected from the epithelium were found to be myosin VIIa positive but negative for p27(Kip1) even though nuclear remnants were still visible. In the transition zone where partial hair-cell loss occurs, freshly ejected hair cells lying immediately above the surface of the BP no longer expressed p27(Kip1). Damaged hair cells within the epithelium in the transition zone contained p27(Kip1) in their cytoplasm but not in their nuclei. These data support recent in vitro findings suggesting that p27(Kip1) protects cells from apoptosis and that its downregulation may be a general feature of programmed cell death.

Sound damage and gentamicin treatment produce different patterns of damage to the efferent innervation of the chick cochlea.

Both sound exposure and gentamicin treatment cause damage to sensory hair cells in the peripheral chick auditory organ, the basilar papilla. This induces a regeneration response which replaces hair cells and restores auditory function. Since functional recovery requires the re-establishment of connections between regenerated hair cells and the central nervous system, we have investigated the effects of sound damage and gentamicin treatment on the neuronal elements within the cochlea. Whole-mount preparations of basilar papillae were labeled with phalloidin to label the actin cytoskeleton and antibodies to neurofilaments, choline acetyltransferase, and synapsin to label neurons; and examined by confocal laser scanning microscopy. When chicks are treated with gentamicin or exposed to acoustic overstimulation, the transverse nerve fibers show no changes from normal cochleae assayed in parallel. Efferent nerve terminals, however, disappear from areas depleted of hair cells following acoustic trauma. In contrast, efferent nerve endings are still present in the areas of hair cell loss following gentamicin treatment, although their morphological appearance is greatly altered. These differences in the response of efferent nerve terminals to sound exposure versus gentamicin treatment may account, at least in part, for the discrepancies reported in the time of recovery of auditory function.

Topically applied diacylglycerols increase pigmentation in guinea pig skin.

Exposure of human and murine melanocytes in vitro to the diacylglycerol (DAG) 1-oleoyl-2-acetyl-sn-glycerol (OAG) markedly increases melanin production within 24 h. To determine whether OAG can increase melanin production in vivo, increasing concentrations of OAG (10-60 mg/ml) in propylene glycol were applied daily for 5 d to shaved guinea pigs. Dose-dependent increased pigmentation was visible first on days 17-22 and persisted for 10-14 weeks. Peak epidermal melanin content in OAG-treated sites was more than twice that of untreated or vehicle-treated sites, as assessed by computerized image analysis of Fontana-Masson stained biopsy cross sections. In another experiment to assess the mechanism of DAG-mediated pigmentation, guinea pigs received twice daily separate applications of OAG, dipalmitoylglycerol (diC16), dioctanoylglycerol (diC8), each 50 mg/ml, 20 microliters/application, and propylene glycol vehicle alone for 5 d. Increased pigmentation was visible after 10 d in the OAG and diC8 sites but not in diC16 or vehicle sites. These results correlate with the reported ability of these compounds to activate protein kinase C in vitro. In a final experiment, guinea pigs received OAG 25 mg/ml three times daily to one test site, and once daily ultraviolet B (70 mJ/cm2, equivalent to 0.6 minimal erythemal dose) radiation to another for 10 d. The OAG and ultraviolet B test sites developed comparable pigmentation by both clinical and histologic criteria. Our data demonstrate that topically applied DAGs can produce a long-lasting increase in epidermal pigmentation, presumably through protein kinase C activation, which clinically and histologically closely resembles ultraviolet-induced tanning.

Migration of hyaline cells into the chick basilar papilla during severe noise damage.

Severe acoustic damage in the chick cochlea causes a destruction of both hair cells and supporting cells in a localized area on the basilar papilla. In this region, the sensory cells are replaced by a layer of flattened epithelial cells. We have employed scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to examine the structure and cytoskeletal changes involved in this process. Immunocytochemical staining for actin indicates that the flattened cells are derived from the hyaline cells normally located along the inferior edge of the basilar papilla. In control cochleae the hyaline cells contain dense bundles of actin filaments that anchor into the basal surface of the cells. The hyaline cells appear to redistribute into the severely damaged region by extending the actin bundles at their basal surfaces. Moreover, the efferent nerves that normally form a network among the hyaline cells move into the severely damaged area along with the hyaline cells. In moderately damaged cochleae, where only hair cells are lost, the hyaline cells do not spread into the damaged region. The functional role of this hyaline cell migration is unknown, but it may be involved in maintenance or repair of the severely damaged cochlea.