Neurotrophins in the Inner Ear

The neurotrophins NT-3 and brain-derived neurotrophic factor (BDNF) are necessary for normal development of the innervation of the inner ear14,27. In mice with constitutive deletion of both NT-3 and BDNF, or their receptors TrkB and TrkC, hair cells develop normally, but their neural connections are dramatically attenuated, both the sensory fibers in the VIIIth nerve and the efferent feedback pathways from the superior olivary complex (for review see19). During inner ear development, NT-3 and BDNF are expressed by supporting cells and hair cells in a decreasing gradient from apex to base13,18. In the postnatal inner ear, both neurotrophins continue to be expressed28,29; however, recent studies of transgenic mice with forced over- and underexpression of NT-3 or BDNF in hair cells or supporting cells suggest that NT-3 expression is more critical for the formation and maintenance of cochlear innervation, while BDNF is more critical for the innervation of the vestibular epithelia17,30.

Prior studies have shown that cochlear perfusion, or round-window delivery, of BDNF and/or NT-3 can prolong the survival of spiral ganglion cells after drug-induced cochlear damage with aminoglycoside antibiotics31,32,33. Furthermore, several studies have documented post-treatment extension of peripheral axons within the osseous spiral lamina and along the denuded basilar membrane with cochlear perfusion of neurotrophins34 or neurotrophin gene delivery via adeno-associated virus vectors35. In these ototoxicity models, the drug dosage was adjusted to produce total hair cell destruction, thus the peripheral processes have no appropriate targets to reconnect with and the spiral ganglion cell survival is functionally important only with implantation of a cochlear prosthesis.

Regeneration vs. Repair of Hair Cells and Synapses

For many years, the dogma in acquired sensorineural hearing loss was that cochlear nerve degeneration only occurred secondarily to hair cell death. Now, we know that if hair cells are destroyed without an external insult, such as noise or ototoxic drugs, cochlear nerve terminals can survive after hair cell loss, so long as supporting cells survive36. Furthermore, in noise-induced1, age-related37 and ototoxic drug damage38, cochlear nerve synapses degenerate in large numbers even when hair cells survive. This paradigm shift, coupled with recent data suggesting that NT3 regulates synaptogenesis on postnatal inner hair cells17, inspired us to ask whether neurotrophin treatments could reverse cochlear synaptopathy and restore cochlear function in cases where the hair cell targets remain intact. The primary degeneration of cochlear nerve synapses will eventually lead to death of the spiral ganglion cells in the absence of intervention1, however, the death of the cell body and central axon takes months to years1, during which time the neurons likely remain electrically excitable, given that cochlear implants continue to function for years after hair cell loss. Thus, there may be a long therapeutic window within which to regenerate the peripheral axons and/or synaptic connections in cases where the hair cells remain intact.

In noise-induced cochlear synaptopathy of the type produced here, confocal analysis shows that loss of pre- and post-synaptic puncta is nearly complete by the end of the exposure22: synaptic counts at 0 hrs are similar to those seen at 24 hrs, 1 wk and 8 wks post exposure1. At even longer post-exposure survivals, the synaptic loss only increases, as age-related changes add to those induced by the noise39. When noise-exposed ears are examined by electron microscopy, immediately after exposure, the inner hair cell area shows swollen cochlear nerve terminals, lacking cytoplasm and with broken membranes40,41. The fact that similar neural ultrastructural damage is after cochlear perfusion of glutamate agonists and that the effect can be blocked with glutamate antagonists, has suggested that noise-induced neuropathy is a type of glutamate excitotoxicity at this highly active synapse42. When cochlear explants are exposed to glutamate agonists in vitro, inner hair cell ribbons quickly disappear and the peripheral terminals of the cochlear nerve retract to the first node of Ranvier, in the osseous spiral lamina43. Thus, the observation of near-normal synaptic counts in some of the high-dose NT-3 ears nine days post-exposure strongly suggests that the treatment has regenerated peripheral terminals and elicited synaptic connections de novo, rather than simply enhancing recovery of damaged neuronal terminals.

In our hands, delivery of the NT-3 treatment yielded bimodal results (Fig. 3B), with roughly a 50% “success rate”. We attribute the failures to the difficulty in delivering a viscous gel through a small micropipette (tip diameter ~ 150 microns), as constrained by the small size of the mouse round window and the small size of the bulla opening required to minimize surgically induced threshold shifts. These constraints forced us to increase poloxamer dilutions to levels that compromised its gelling properties and likely decreased the contact time of the injectate with the round window membrane as it became diluted with exudate from the surrounding tissue and diffused away from the injection site. This, in turn, likely decreased the effective dose of NT-3 delivered to the cochlear fluids.

The special vulnerability of hair cells in the basal tip, or “hook” of the cochlear spiral (Fig. 1) has been observed after acoustic overexposure in chinchilla, cat, guinea pig and mouse9,44,45,46. The phenomenon is not well understood: for example, it cannot be explained based on measurements of cochlear mechanical motion47. The NT-3 mediated rescue of the noise-induced OHC lesions in the hook (Fig. 1F) is consistent with prior reports48,49. Studies of the dynamics of hair cell death after noise suggest that, for exposures similar in severity to those used here, there is significant ongoing hair cell death between 24 hrs and 14 days post-exposure46. Considering that NT-3 has been reported to have protective effects on hair cell survival48,49 (and that there is no evidence suggesting a role for NT-3 in hair cell regeneration), the beneficial effects of NT-3 delivery at 24 hrs post exposure likely represent enhanced recovery of damaged hair cells rather than regeneration of missing hair cells. Given that, adult hair cells can survive indefinitely after complete cochlear nerve degeneration subsequent to central axotomy50, the hair cell rescue is likely a direct effect of the NT-3, rather than an indirect effect through enhanced synaptic connection. However, the mechanism is unclear, since in the adult cochlea only spiral ganglion neurons and their processes express Trk receptors19.

Application to Human Hearing Impairment

Both hair cells and cochlear nerve fibers are required for normal hearing. Damage to hair cells elevates the sound pressure required for stimulus detection. This “threshold shift” is well measured by conventional pure-tone audiometry and is what has conventionally been thought of “hearing loss”. Damage to cochlear nerve fibers, per se, will not elevate audiometric thresholds unless the loss is catastrophic, i.e. >90%51,52); however, the loss of functional neural channels will reduce the intelligibility of complex sounds like speech, even when they are detectable. Complementing the animal work showing that cochlear synapses are the most vulnerable elements in noise-damage1, aging37 and aminoglycoside otototoxicity38, a recent study of aging humans suggests that loss of synaptic connections between spiral ganglion cells and surviving hair cells is an important aspect of presbycusis53 and that this cochlear neuropathy may be a major cause of the most common hearing-related complaint of the elderly, i.e. difficulties understanding speech in a noisy environment54.

The idea that a significant, but ultimately reversible, noise-induced threshold shift can be associated with a 50% loss of cochlear nerve synapses has also provided a new hypothesis for the origins of tinnitus, the phantom perception of sound that is often the permanent result of a noise exposure even if the threshold elevation is transient55. It has been argued that the permanent loss of spontaneous and sound-driven activity in a subgroup of cochlear nerve fibers leads to a central gain readjustment that drives hyperactivity in central auditory pathways and thereby causes tinnitus5.

Thus, there is likely a significant clinical population with some degree of hearing impairment (or tinnitus) that could be addressed by a treatment analogous that used here in noise-exposed mice56. The present experiments show that round window delivery can be effective local access route to the inner ear, even for a protein as large as NT-3 (13.6 kD molecular weight). Here, we used a poloxamer, a thermoreversible hydrogel that is liquid at 4 °C but gels at body temperature, to extend the drug delivery time57, which is important for a molecule like NT-3 with a short half-life once it enters body fluids58. Prior studies have seen biological effects of neurotrophins delivered to the round window using a variety of slow-release strategies59.

Although the success of local NT-3 delivery in regenerating cochlear synapses when administered 24 hrs post exposure is an important proof of concept, many key questions remain, most importantly how long after the insult can neurite extension and synaptogenesis still be elicited.