We used Pou4f3 mutant mice as a model for testing whether neurotrophin treatment can induce nerve fiber regeneration and SGN preservation in ears with hereditary deafness. In untreated Pou4f3 homozygotes at 6 weeks of age, the number of peripheral nerve fibers and the density of SGNs were much lower than in normal mice. Ad.BDNF inoculations into the cochlear fluids of these mutant mice lead to additional growth of nerve fibers into the auditory epithelium and preservation of SGNs in Rosenthal's canal.
mutant mouse serves as a model of hereditary hearing loss. Pou4f3
homozygotes are completely deaf because cochlear hair cells fail to develop31,34,35
. The normal organization of supporting cells is also absent. Instead, the auditory epithelium appears nearly flat, with the exception of one type of cell, an AR cell based on its appearance in phalloidin-stained whole-mounts. We demonstrate that clusters of AR cells are scattered periodically along the cochlear duct. The identity of the AR cells is difficult to determine. Given the lack of signals from adjacent hair cells, it is possible that the AR cells do not express pillar cell or Deiters cell markers, or might even mis-express them. We could not detect other marker proteins, such as p75 in Pou4f3
-null ears (data not shown). Based on their location, AR cells most likely represent the same cells previously referred to as “pillars”36
. Although AR cells are not a typical or general finding in ears with early hair cell loss37
, similar clustering of pillar cells has been observed in other deaf ears that had an Atoh1 conditional deletion38
. Together, these two mutations may provide insight into the mechanism of pillar cell formation in the absence of hair cells. Studies of the Atoh1 conditional deletion animals indicated that FGF8 may signal development of pillar cells via the Fgfr3 receptor39,40
, and our results indicate that this cellular regulation occurs in the absence of differentiated hair cells.
Toluidine blue-stained cross-sections of the Pou4f3 ears revealed that AR cells had a dark cytoplasm, compared to neighboring supporting cells, and were closely associated with each other within a cluster. Cells in the space between clusters of AR cells resembled a flat epithelium. The nuclei of AR cells were more oval than round, whereas most other supporting cells in the auditory epithelium have a round nucleus, including pillar cells in wild-type ears.
Sugawara et al.10
previously described the relationship between supporting cell survival and neuronal survival and postulated that pillar cells may be at least as important as hair cells for the purpose of maintenance of nerve survival10,20
. Our data generated using a mouse model of a mutation that affects hair cell differentiation and survival support the findings of Sugawara et al.10
. The mechanism by which supporting cells maintain nerve fibers is not completely clear, but a likely explanation involves an elevated level of neurotrophins expressed by these cells13,19
. The ability of nerve fibers to sprout in response to neurotrophins has been shown in deaf ears of guinea pigs25,41,42
, and now we demonstrate similar findings in the mouse. While the AR cells (regardless of their identity, pillar and/or Deiters) appear to play a role in attracting a few fibers in mutant ears without neurotrophin treatment, the Ad.BDNF
treatment appears to dramatically increase both the number of nerve fibers in the auditory epithelium and the survival of the auditory neurons in Rosenthal's canal.
Our results showed that in mutant cochleae treated with Ad.BDNF
, the sprouting of nerve fibers was significantly greater in the apical turn than in the basal turn. This was surprising because the inoculation of the viral vector was into the basal turn, likely yielding a higher concentration of BDNF in the base. There are several possible explanations for this unexpected finding. First, SGN survival in the apical cochlea was better than in the base in both control and treated ears, providing more neurons for sprouting. In addition, it is possible that apical neurons are more responsive to BDNF than basal neurons. This would be in agreement with the developmental role of BDNF in determining the innervation pattern of the cochlea, where BDNF plays a greater role in the apical turn than in the base12
. Finally, it is possible that the concentration of BDNF in the apex was lower, but closer to normal physiological levels than in the base, and that excess neurotrophin is not necessarily better for nerve fiber sprouting. Because the rate of viral vector-mediated transgene expression is unregulated, it is possible that the concentration of neurotrophins attained in the cochlea is higher than normal.
Because the type I afferents are the relevant population of neurons for receiving cochlear implant stimulation and transmitting electrically evoked signals to the brain stem, it is important to identify the fibers that grow into the auditory epithelium following neurotrophin transgene expression. Previous studies examined resprouting nerve fibers in ears treated with BDNF and acidic FGF and identified them as afferent nerve fibers41,42
. In addition, experiments involving transgenic mice expressing BDNF
have indicated that the neurotrophin is the most important molecule for inner ear afferent fiber guidance43,44,45
. In the current study, both narrow and thick nerve fibers were observed growing into the auditory epithelium after Ad.BDNF
inoculation. This result suggests that transgenic BDNF
expression in the area of the auditory epithelium may induce growth of more than one population of neurons. Possibilities include both Type I and Type II afferent dendrites as well as olivocochlear efferent nerve fibers. Developing Type II afferent fibers have recently been shown to respond preferentially to BDNF in vitro46
. Another explanation for the presence of thick and thin fibers is that some afferent fibers may respond to the BDNF treatment by changing their diameter. Better characterization of the type of fibers that sprout into the deaf ear after BDNF treatment will need to be performed in future studies.
We observed several fibers that exhibited localized swelling, most commonly near the medial aspect of the AR cells. These swellings sometimes appear as an enlarged nerve ending, and other times as an enlarged region away from the ending. Previous reports have described similar varicosities in guinea pig SGN fibers treated with neurotrophins25,41
. There are other examples of bulging areas in neurons47,48
and their nature is unclear. One possible explanation provided by Lee et al.48
is that enlarged regions serve to orient neurons in the tissue, but it is also possible that these enlarged endings are associated with stress or a constant search for a target for synaptogenesis. Alternatively it has been proposed that such varicosities form whenever anterograde transport of organelles and other growth cone substrates temporarily exceed the rate of growth cone extension49
The degeneration of SGNs in Pou4f3
mutant mice has been described31
. Here we show that inoculation with Ad.BDNF
resulted in the maintenance of a significantly higher density of SGNs in Pou4f3
mutant mouse ears. To our knowledge this is the first demonstration of the ability to prevent a loss of inner ear neurons due to hereditary disease. The induced neurotrophin expression most likely had several parallel effects, inducing nerve fiber extension, preventing degeneration of the SGN somata and increasing cell size. BDNF-induced survival of the SGNs was more effective in the apical turn than in the base, although the density of SGNs in Rosenthal's canal in the hook was higher than in the base. BDNF also increased SGN somata size only in the apex. We can only speculate on the reason for this surprising pattern. One possibility is that the inoculation caused mechanical trauma in the base. Another possibility is that the concentration of BDNF in the base, close to the inoculation site, was excessive, whereas the hook and the apex, flanking the base on each side, received a concentration of the neurotrophin that is closer to optimal. The fact that the nerve fiber regeneration was relatively less pronounced in the base (compared with the apex) may also be related to the finding that the density of SGNs in the base was the lowest in the cochlea (lower than the hook and much lower than the apex).
Potential negative side effects must be considered in the development of any clinical applications of our work. We noted that two weeks after Ad.BDNF
inoculation, connective tissue could be observed in the scala tympani. Prior studies have shown similar findings following viral vector inoculation25
or exogenous BDNF inoculation and electrode stimulation50
. Growth of fibrous tissue in the cochlear fluid spaces is an undesirable side effect that may lead to several negative outcomes, including an increase in electrode impedance51
and difficulty in re-insertion of an electrode if revision surgery becomes necessary. It is unclear how elevated levels of BDNF in endolymph (following inoculation into the scala media) causes changes in cells lining the scala tympani. However, BDNF is a pleiotropic factor that can originate from more than one source and influence diverse cell types. It is expressed by inflammatory cells including macrophages and T cells52
, and in the lung it can be associated with airway obstruction53
. Once the dynamics of neurotrophin diffusion in the cochlear fluids are better understood, it will also be possible to design a strategy to prevent or limit this side effect. A possible solution is to lower the viral titer to reduce neurotrophin levels, assuming a lower concentration of neurotrophins decreases connective tissue growth. Alternatively, a specific reagent that can antagonize connective tissue growth could be added at the time of the neurotrophin therapy. Other methods for gene delivery could also be considered, as previously shown with electrode coatings with allogeneic flbroblasts54
or using alginate capsules55
. Due to the minute size of the mouse ear and correspondingly small volume of its cochlear fluids, accurate measurements of concentrations of neurotrophins in the ear cannot be accomplished but work on larger animals may help correlate gene delivery methods with resulting neurotrophin concentrations, to advance these methods towards clinical applications.
Among the advantages of adenoviral vectors are their ability to infect a broad range of cell types with high efficiency, and the rapid onset of gene expression following infection. However, adenovirus gene expression is transient, and therefore elevated levels of transgenes cannot be sustained over several weeks. Therefore, the data we present can serve as a proof for the principle that nerve fibers can be regenerated in the mutant ear, but for future clinical application, it would be better to use a long-term expressing vector such as adeno-associated virus which can sustain gene expression for a long time56
In many human deafness cases, SGNs survive for many years, despite the loss of most hair cells and the degeneration of peripheral nerve fibers from the auditory epithelium5
. This finding is not limited to environmental hair cell loss, but also holds for many of the mutation-caused hereditary deafness cases. However, there are mutations in which spiral ganglion neurons do not survive, and these patients could benefit from a treatment for enhancing neuronal survival. One important example is neurofibromatosis 2 (NF2) which involves severe degeneration of SGNs. In many of the NF2 patients SGN degeneration makes the cochlear implant impractical leaving a brainstem implant as the only therapeutic option. When a small number of SGNs survive, a traditional cochlear implant is of some benefit57
, suggesting that any therapy to enhance preservation of the neurons would further improve the cochlear implant therapy outcome. There are other mutations with variable outcome of cochlear implant therapy, such as DFN3, where the variable outcome is not yet understood58
. It is possible that enhancing preservation of SGNs would increase the success of the cochlear implant procedure, especially in patients who otherwise do poorly with their implant. Even in cases where SGNs survive in human cochleae for a long time, cochlear implant patients may sometimes do very poorly with their prosthesis59
. It is possible that inducing auditory nerve fiber regeneration in such ears could improve the outcome of the cochlear implant therapy. The concept relating the cochlear substrate with cochlear implant outcome has been demonstrated60,61,62
. In addition, transgenic BDNF
over-expression with concurrent electrical stimulation has been shown to improve cochlear implant thresholds and survival of auditory neurons63
The data we report, showing substantial neural preservation and regeneration in a mouse model for human hereditary deafness, join other recent advances in restoring structure and function of ears with hereditary disease64,65
. These advances provide milestones in the progress towards treating humans who suffer from severe hereditary inner ear disease. We provide a proof of principle that ears affected by genetic diseases that do not target neurons directly, are amenable to treatment aimed at enhancing nerve survival, thereby providing an avenue for enhancing cochlear implant outcomes in patients with hereditary hearing loss. In addition, improving nerve survival and fiber regeneration will also augment the outcome of future hair cell replacement therapies based on transdifferentiation or stem cell transplantation.