T. R. Van de Water

NT-3 and/or BDNF therapy prevents loss of auditory neurons following loss of hair cells.

Destruction of auditory hair cells results in a subsequent loss of auditory neurons. In situ hybridization and neuronal cell culture studies as well as analyses of the inner ears of neurotrophin and neurotrophin receptor gene knockout mice have shown that NT-3 and BDNF mediate both the development and survival of auditory neurons. In this study guinea pigs were exposed to the ototoxic combination of an aminoglycoside antibiotic and a loop diuretic and then received 8 weeks of intracochlear infusion of either NT-3, BDNF or NT-3 + BDNF to determine whether site-specific application of these neurotrophins could prevent the loss of auditory neurons that follows a loss of auditory hair cells. Infusion of either NT-3 or NT-3 + BDNF into the scala tympani resulted in a > 90% survival of auditory neurons while BDNF infusion yielded a 78% survival rate, compared with a 14–24% neuronal survival rate in untreated ototoxin-exposed cochleae. These results show that loss of auditory neurons that occurs subsequent to a loss of auditory hair cells can be prevented by in vivo neurotrophin therapy with either NT-3 or BDNF.

Caspase inhibitors prevent cisplatin-induced apoptosis of auditory sensory cells

IN VITRO studies tested the efficacy of three caspase inhibitors, Ac-VAD-cmk (caspase-1 inhibitor), z-DEVD-fmk (caspase −3 inhibitor) and B-D-fmk (BOCDFK, a general inhibitor), for protecting auditory sensory cells from cisplatin-damage induced loss. Treatment of 3-day-old rat organ of Corti explants with these caspase inhibitors protected > 80% of the auditory hair cells from cisplatin-damage initiated apoptosis. Dissociated cell cultures of 3-day-old rat spinal ganglia treated with any of these three caspase inhibitors in addition to exogenous neurotrophin have highly significant increases in neuronal survival following cisplatin exposure. These results indicate that loss of auditory sensory cells as a result of cisplatin-induced damage involves apoptosis and that blocking of this cell death pathway at the caspase level effectively rescues both hair cells and neurons.

Organ Culture of the Mammalian Inner Ear

An organ culture technique that is 90% effective has been established that will routinely allow the growth and development of the twelfth gestation day mouse inner ear. The in vitro development lags behind the in vivo development but follows the pattern of in vivo development.

Neurotrophins affect survival and neuritogenesis by adult injured auditory neurons in vitro.

This study evaluates the trophic effects of three neurotrophins on traumatized adult auditory neurons in culture, and the presence of these neurotrophins in cochlear nucleus tissue. BDNF and NT-3 promoted survival but very limited neuritogenesis by adult auditory neurons in vitro, while NGF, although without a survival effect, evoked a robust neuritic outgrowth response when combined with BDNF. Messenger RNAs that encode for NGF, BDNF and NT-3 were detected by RT-PCR in RNA extracts from adult cochlear nuclei tissue. Based on these in vitro and in vivo findings, we propose NT-3 as the agent of the peripheral target-derived survival promoting effect and NGF, BDNF, and NT-3 as mediators of trophic influences originating from the central target (i.e. cochlear nucleus).

Protection of Both Auditory Hair Cells and Auditory Neurons from Cisplatin Induced Damage

Cisplatin is an effective anti-neoplastic agent used in the treatment of squamous cell cancer of the head and neck, but with serious side effects. One serious side effect is damage to both the auditory hair cells and the auditory neurons. The damage to the neurons has been shown to be a direct effect and not due to the loss of the neurotrophic support provided by the hair cells. Several neurotrophins have been shown to lessen the extent of cisplatin induced damage of auditory neurons in vitro, but these neurotrophins have had no effect on the extent of damage to the hair cells. D-methionine (D-met) has been demonstrated to provide protection against cisplatins nephrotoxicity in vivo and ototoxicity in vitro. In this study the combination of brain derived neurotrophic factor (BDNF) with D-met has shown that both auditory neurons and auditory hair cells can be protected from cisplatin induced damage in vitro. These results demonstrate that this type of combination therapy (i.e. a neurotrophin combined with a free radical scavenger) can provide more complete protection for the auditory receptor against cisplatin toxicity than either of these agents alone. Because both BDNF and D-met have been shown to have trophic activity in vitro we proposed that the combination of these agents will also provide effective protection against cisplatin induced ototoxicity and neurotoxicity of the auditory receptor in vivo.

NEUROTROPHIC EFFECTS OF BDNF AND CNTF, ALONE AND IN COMBINATION, ON POSTNATAL DAY 5 RAT ACOUSTIC GANGLION NEURONS

The neuronal survival promoting ability of brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), individually and in combination, was evaluated in dissociated cell cultures of postnatal day 5 (P5) rat acoustic ganglia. The neuritogenic promoting effect of these same neurotrophic factors was examined in organotypic explants of P5 rat acoustic ganglia. The results showed that BDNF was maximally effective at a concentration of 10 ng/mL in promoting both survival and neuritogenesis of these postnatal auditory neurons in vitro. CNTF was maximally effective at a concentration of 0.01 ng/mL at promoting both survival and neuritogenesis in the acoustic ganglion cultures. BDNF had its strongest effect on neuronal survival while CNTF was most effective in stimulating neurite outgrowth. These two neurotrophic factors, when added together at their respective maximally effective concentrations, behave in an additive manner for promoting both survival and neuritic outgrowth by the auditory neurons.

BDNF protection of auditory neurons from cisplatin involves changes in intracellular levels of both reactive oxygen species and glutathione

Previous studies have shown that brain derived neurotrophic factor (BDNF) can protect auditory neurons from cisplatin toxicity in vitro. To explore the mechanism of BDNF mediated neuronal protection sequential confocal microscopic sampling of auditory neurons measured intracellular levels of reactive oxygen species (ROS) in response to withdrawal of BDNF supplementation, cisplatin exposure, and BDNF protection from cisplatin damage in normal and oxidative stress states. Additionally, we examined intraneuronal levels of the free radical scavenger glutathione (GSH) in response to withdrawal of BDNF. Withdrawal of BDNF resulted in increased production of ROS and decreased survival of auditory neurons. Levels of GSH within neurons increased after BDNF withdrawal, and this increase was shown to lag behind the production of ROS. Auditory neurons in cultures supplemented with BDNF and exposed to cisplatin showed significantly lower levels of ROS and increased survival compared to neurons in unprotected, cisplatin exposed cultures. Neurons treated with buthionine sulfoximine (an inhibitor of GSH synthesis), supplemented with BDNF, and exposed to cisplatin showed significantly higher intracellular levels of ROS compared to the neurons in BDNF supplemented cultures exposed to cisplatin. These results suggest that intracellular levels of ROS play an important role in cisplatin induced cell death of auditory neurons and that production of ROS can be ameliorated through supplementation with BDNF. GSH appears to mediate BDNF protection of these neurons from cisplatin induced ROS and subsequent damage.

Effects of neurotrophins on early auditory neurones in cell culture.

During the first week of postnatal development, the innervation of the organ of Corti changes from an immature to an adult pattern. Dissociated cell cultures of early postnatal spiral ganglia were used to investigate the effects of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on maturing auditory neurones. BDNF was the most potent stimulator of neuritogenesis, NT-3 provided the strongest support for neuronal survival, while NGF supported limited neuritogenesis, and only at pharmacological levels. These findings suggest that both BDNF and NT-3 participate in the postnatal maturation of cochlear innervation and that NGF is most probably not involved in this process.

NT-3 combined with CNTF promotes survival of neurons in modiolus-spiral ganglion explants

AUDITORY neurons depend upon the integrity of both their peripheral (auditory hair cells) and central (cochlear nucleus) targets for survival. One proposed trophic mechanism is the production of neurotrophin-3 (NT-3) by auditory hair cells. Modiolus-spiral ganglion explants from adult rats that closely mirror cell-cell interactions and in vivo tissue relationships within this ganglion provide a model for testing trophic factors. Brain derived neurotrophic factor (BDNF), NT-3 and ciliary neuro-trophic factor (CNTF) were tested for their ability, both individually and in combination, to support neuronal survival. NT-3 was the strongest individual promoter of survival, while CNTF (a cytokine) with NT-3 (a neurotrophin) was the most effective combination for promoting the survival of auditory neurons.

Growth Factor Interactions in Cultures of Dissociated Adult Acoustic Ganglia: Neuronotrophic Effects

Auditory neurons cultured from adult rat acoustic ganglia require for survival either a substrate bound factor(s) present in astrocyte conditioned medium or substrate bound basic fibroblast growth factor (bFGF). Nerve growth factor (NGF) is not a survival factor for these neurons in vitro, but when used in combination with substrate bound bFGF, NGF does vigorously stimulate a neuritogenesis response by these neurons. Transforming growth factor beta (TGF beta 1) enhances the survival effect that bFGF has on these adult auditory neurons but does not by itself promote their survival in dissociated acoustic ganglion cultures. We propose that there may be complex interactions and synergy exerted by these growth factors (i.e. bFGF, NGF, TGF beta 1) during injury to the inner ear.