A single dose of neurotrophin-3 to the cochlea surrounds spiral ganglion neurons and provides trophic support
Introduction
Neurotrophins are a group of proteins that promote neural differentiation, survival and neurite outgrowth. The neurotrophin family includes nerve growth factor (NGF), neurotrophin-3 (NT3), neurotrophin-4/5 (NT4/5) and brain-derived neurotrophic factor (BDNF). Neurotrophins bind and activate tropomyosin related kinase (Trk) receptors. The principal but not exclusive binding relationships are NGF to TrkA, BDNF and NT4/5 to TrkB and NT3 to TrkC. All four neurotrophins also bind with low affinity to the p75 neurotrophin receptor (p75NTR) (Lee et al., 2001).
Sensory hair cells of the cochlea express and release BDNF and NT3, while spiral ganglion neurons (SGNs) that terminate at the hair cells express Trk receptors (Ylikoski et al., 1993). There is evidence that BDNF and NT3 released by hair cells maintain the health and connections of SGNs. This is supported by the fact that, following an ototoxic insult in which hair cells are permanently lost, there is progressive degeneration of the peripheral processes of SGNs over the course of eight weeks in guinea pigs, and eventually apoptosis of the SGNs (Terayama et al., 1977).
Apoptosis of SGNs following ototoxin exposure in guinea pigs is preventable by long-term administration of exogenous neurotrophins to the scala tympani. When given prior to or simultaneous to deafening, there is up to 95% survival of SGNs and hair cell protection (Ernfors et al., 1996, Ruan et al., 1999, Yagi et al., 1999, Suzuki et al., 2000). When given 4–7 days after deafening, 78–100% of SGNs survive (Staecker et al., 1996, Staecker et al., 1998, Miller et al., 1997, Yagi et al., 2000, Shinohara et al., 2002, Gillespie et al., 2003). In addition to SGN survival, there is also evidence that the peripheral processes regenerate in response to neurotrophins (Staecker et al., 1996, Miller et al., 1997, Wise et al., in press). The promising results from these studies suggest that cochlear implant patients can benefit from neurotrophin treatment by maintaining a denser population of SGNs and peripheral processes and improving the nerve excitation capability of the cochlear implant.
It is postulated that neurotrophins may be clinically useful during cochlear implantation or other surgical procedures on the inner ear to protect the neurons from trauma or ototoxicity. Experiments to date have utilized a continuous neurotrophin delivery system consisting of a mini-osmotic pump and cannula that risk introducing infection into the cochlea and would be difficult to manage in the clinical situation. To inject a single dose of neurotrophins into the cochlea at the time of a surgical procedure would be simple and convenient. This preliminary study will assess the feasibility of such a treatment, determining the effects of a single dose of NT3 on SGN health and survival compared to continuous infusion. In addition, the relative amounts of neurotrophin that reach each part of the cochlea will be examined using 125I-labelled NT3.
The 125I label is a well documented research tool and has been used to trace retrograde transport of NT3 in sympathetic and sensory neurons (von Bartheld et al., 1996, Reynolds and Hendry, 1999, Steljes et al., 1999). The 125I label does not affect the ability of NT3 to undergo retrograde transport nor its ability to promote survival in auditory neurons (Reynolds and Hendry, 1999, Richardson et al., 2004). It was previously shown that a single infusion of 125I NT3 into the cochlea results in a broad distribution throughout cochlear tissues, with a minor proportion reaching SGNs (Richardson et al., 2004). In this study, increased concentrations of 125I NT3 were infused into deafened guinea pigs, enabling measurements of trophic effects on SGNs to be compared to the diffusion and relative dose of 125I NT3 in each part of the cochlea.
Section snippets
Animals
Adult pigmented Dunkin–Hartley guinea pigs averaging 455 g were bred within the Department of Otolaryngology of the University of Melbourne. A total of 20 guinea pigs are described in this study. Time-mated pregnant Albino–Wistar rats were purchased from the University of Adelaide. The Animal Research Ethics Committee of the Royal Victorian Eye and Ear Hospital approved the care and use of the animals.
NT3 labelling and batch testing
10 μg human recombinant NT3 (Chemicon International, USA) was labelled with 0.75 mCi Na 125I (ICN
Guinea pig deafening
All 20 guinea pigs used in this study had hearing thresholds within the normal hearing range (33–48 dB SPL). Four were used as normal-hearing controls and infused with a single pulse of Ringers solution (Group 1). The remainder were deafened for a period of 28 days prior to commencement of treatment, resulting in threshold shifts of 55 dB SPL or more. Treatment was either infusion of a single pulse of Ringers solution, 100 ng 125I NT3 or 140 ng 125I NT3 with a 7-day survival period (Groups 2–4,
Degenerative effects of deafening
Deafening with aminoglycosides produced substantial degeneration of the organ of Corti and SGNs after 35 and 56 days, with the basal turn most severely affected (Fig. 1). These results concur with those previously reported for guinea pigs in which 73% and 46% of SGNs survive after 33 and 61 days of deafening, respectively, and also mimic the clinical situation in which a proportion of SGNs are lost after varying causes of deafness (Nadol, 1997, Gillespie et al., 2003, Wise et al., in press).
Effects of 125I NT3 on SGNs
Conclusions
Infusion of neurotrophin into the scala tympani results in a broad distribution into neural and non-neural tissues of the cochlea, although diffusion does not always extend to the apical turns. The peripheral processes and the cell bodies of SGNs become bathed in neurotrophin, although 125I NT3 has not been detected within SGN cell bodies to date. The proportion of neurotrophin that reaches the peripheral processes and SGN cell bodies is low compared to other tissues in the basal turn. This
Acknowledgements
The authors acknowledge the help and expertise of Maria Clarke and Prudence Nielsen for histological procedures conducted in this study. The Stavros S. Niarchos Foundation generously supports this work.
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