Учебники / Auditory Trauma, Protection, and Repair Fay 2008

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may persist in the mature cochlea (Wiechers et al. 1999; Hansen et al. 2001b; Zha et al. 2001).

NT-3 is initially expressed throughout the organ of Corti sensory epithelium in mammals but during embryonic development becomes more restricted. Expression of NT-3 early in development is absolutely required for survival of SGNs: most SGNs die during embryogenesis in knockout mice lacking NT-3 or TrkC (Fritzsch et al. 2004). In the postnatal rodent organ of Corti, NT-3 is expressed only in the inner hair cells and adjacent supporting cells (Sugawara et al. 2007).

In addition to the neurotrophins, there are other families of peptide factors in which some or all members are neurotrophic. One such peptide neurotrophic factor, GDNF, appears to be synthesized in the organ of Corti, starting in the second postnatal week, and can support SGN survival in vitro and in vivo (Ylikoski et al. 1998; Yagi et al. 2001).

There has been no test of the postnatal requirement for hair cell–derived neurotrophic factors, such as NT-3 and GDNF, for SGN survival. Mice lacking these factors either lose their SGNs before birth in the case of NT-3 or die before birth in the case of GDNF. It is possible that given physiological electrical stimulation via normal afferent input, SGNs may be able to survive in the absence of hair cell–derived neurotrophic factors. Also, it should be noted that neurotrophic factors have roles other than survival, including synaptic maintenance and function and control of the mature neuronal phenotype. For example, BDNF and NT-3 have been implicated in inducing, respectively, the characteristically basal and apical physiological properties of SGNs including ion channel composition and firing pattern (Adamson et al. 2002; Zhou et al. 2005). Targeted and conditional gene deletion will be necessary to resolve the role of neurotrophic factors in SGN survival and function in the postnatal cochlea.

5.2.2 Support of SGNs by Multiple Neurotrophic Factors In Vitro

As noted earlier, BDNF, NT-3, or GDNF can each support survival of SGNs in culture (Lefebvre et al. 1994; Hegarty et al. 1997; Ylikoski et al. 1998). Combining neurotrophins BDNF and NT-3 results in increased SGN survival relative to individual neurotrophins (Lefebvre et al. 1994; Hegarty et al. 1997). Moreover, combining neurotrophins with other peptide neurotrophic factors, FGF, Leukemia Inhibitory Factor (LIF), transforming growth factor- (TGF-), or ciliary neurotrophic factor (CNTF) also increases survival relative to neurotrophin alone (Hartnick et al. 1996; Marzella et al. 1997, 1998). In addition, these factors promote neurite growth from cultured SGNs (Lefebvre et al. 1994; Hegarty et al. 1997).

5.2.3 Receptors for Peptide Neurotrophic Factors

5.2.3.1 Neurotrophins and Trks

Receptors for most peptide neurotrophic factors are either receptor proteintyrosine kinases or associate with and activate protein-tyrosine kinases. Of the

neurotrophic factors, the most widespread in the nervous system and best studied are the four members of the neurotrophin family, NGF, BDNF, NT-3, and NT-4 (Huang and Reichardt 2001). The cognate family of receptor protein-tyrosine kinases is the tropomyosin-related kinase (Trk) family which consists of three members, TrkA, TrkB, and TrkC. NGF binds and signals via TrkA receptors, BDNF and NT-4 via TrkB receptors; NT-3 acts principally via TrkC receptors but NT-3 can bind and signal via both TrkA and TrkB receptors (Huang and Reichardt 2003).

5.2.3.2 Neurotrophin Receptor p75NTR

In addition to binding and signaling via Trk receptors, all four neurotrophins, and their unprocessed precursors, bind and signal through an unrelated receptor, the neurotrophin receptor p75NTR (Huang and Reichardt 2003; Gentry et al. 2004). p75NTR is not a protein-tyrosine kinase but does interact with Trk receptors, with the consequence that these receptors reciprocally modulate each other’s signaling. p75NTR also initiates several pathways that activate NF- B, and, importantly, proapoptotic signaling, including JNK. p75NTR is expressed in the developing cochlea in SGNs and in non-neuronal cells (von Bartheld et al. 1991; Gestwa et al. 1999). In the mature cochlea, p75NTR is at low or undetectable levels but is strongly upregulated in the spiral ganglion following hair cell loss (Tan and Shepherd 2006) and so may contribute to SGN degeneration. Mice with mutant p75NTR have apparently normal cochlear development and function but lose hair cells and SGNs at an accelerated rate as they age (Sato et al. 2006). It remains to be established whether the loss of the sensory and neural elements is due directly to loss of p75NTR function in these cells or is an indirect result of loss of p75NTR function in non-neuronal cells.

5.2.3.3 GDNF Family Receptors

The GDNF subfamily of the TGFfamily of growth factors, GDNF, neurturin, artemin, and persephin (Baloh et al. 2000), all bind and signal via the Ret receptor protein-tyrosine kinase or by binding to the neuronal cell adhesion molecule (NCAM; Paratcha et al. 2003). For high-affinity binding an additional “coreceptor” is required: a member of the GDNF family receptor (GFR ) family. This family has four members, GFR 1–4, which are the cognate coreceptors for the four GDNF family ligands. As noted in the preceding text, SGNs express GDNF receptors and GDNF is a neurotrophic factor for SGNs.

5.2.4 Neurotrophic Factor Signal Transduction and Prosurvival Intracellular

Signaling Pathways

Trks and Ret, receptors for neurotrophins and GDNF-family ligands, respectively, are receptor protein-tyrosine kinases. For neurotrophins, activation of the receptor protein-tyrosine kinase appears to be sufficient for initiation of intracellular signaling. Signal transduction for GDNF-family ligands is more complex. Whether the ligand binds to Ret or to NCAM, recruitment of other proteintyrosine kinases by the receptor is additionally required (Encinas et al. 2001;

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Paratcha et al. 2003). In all cases, understanding receptor protein-tyrosine kinase signal transduction is fundamental to understanding how neurotrophins, GDNF, and other peptide neurotrophic factors can initiate prosurvival intracellular signaling.

The signal transduction mechanisms for receptor protein-tyrosine kinases and, in particular, for Trks, have been extensively reviewed (Huang and Reichardt 2003). In brief, binding of ligand to the receptor results in dimerization, cross-phosphorylation of the dimerized protein-tyrosine kinases, and assembly of protein complexes containing adaptor and signal transducer proteins that initiate the various pathways.

One intracellular signaling pathway is initiated by activation of the lipidmodifying enzyme phosphatidylinositol 3-OH kinase (PI3K), which leads to recruitment and activation at the plasma membrane of the protein kinase PKB/Akt, crucial prosurvival kinase for promotion of survival by peptide neurotrophic factors (reviewed in Datta et al. 1999). Inhibition of PKB abolishes the survival-promoting effect of BDNF or NT-3 on SGNs in vitro (Hansen et al. 2001b). PKB promotes survival via phosphorylation of multiple targets (Datta et al. 1999). As noted earlier, PKB inhibits apoptosis by phosphorylating and inactivating forkhead transcription factors (Brunet et al. 1999) and by phosphorylating and inactivating the proapoptotic Bcl-2 family protein Bad (Downward 1999).

Another neurotrophic factor–activated intracellular pathway important for survival is the ERK subfamily of the MAPKs. ERKs promote survival by phosphorylating diverse targets. ERK appears to directly phosphorylate Bad, inactivating this proapoptotic effector (Downward 1999). Another outcome of ERK signaling is activation of the prosurvival transcription factor CREB in the nucleus (Dawson and Ginty 2002).

5.3 Support of SGN Survival by Electrical Activity/Membrane Depolarization

Electrical activity, specifically depolarization, is a survival-promoting stimulus for neurons, principally due to increased cytosolic Ca2+ (Franklin and Johnson 1994; Green 2000). In particular, for SGNs, membrane electrical activity provides a prosurvival stimulus, evidenced by experiments on chronically depolarized SGNs in vitro (Hegarty et al. 1997) and electrically stimulated SGNs in vivo (reviewed in Miller 2001). This raises the possibility that electrical stimulation provided by a cochlear implant, alone or in combination with other therapy, in supporting survival of SGNs, further discussed later.

5.3.1 Membrane Depolarization Supports Neuronal Survival Via Ca2+

Signaling

Multiple intracellular signaling pathways link membrane depolarization to survival (Hansen et al. 2001b), although there may be variability among neurons

with regard to the relative importance of different pathways. A common element among these intracellular signaling pathways is that they are initiated by entry of Ca2+ ions into the cytosol via voltage-gated Ca2+ channels (VGCCs). In neurons chronically depolarized by elevating extracellular K+, Ca2+ entry via L-type VGCCs is required for neuronal survival (Franklin et al. 1995; Galli et al. 1995). It has been suggested that Ca2+ entry through other VGCCs, e.g., N- type, may be more important when neurons are stimulated by patterned electrical activity (Brosenitsch and Katz 2001) in vitro; nevertheless, support of SGNs in vivo by patterned electrical stimulation is inhibited by blockade of L-type Ca2+ channels (Miller et al. 2003), similar to what was observed with SGNs chronically depolarized in vitro (Hegarty et al. 1997).

It may seem paradoxical that cytosolic Ca2+ acts as a prosurvival signal, given that it has been long known that high [Ca2+]i results in excitotoxic cell death (see Section 6). The resolution of this paradox lies in the concentration dependence of the consequences of cytosolic Ca2+. Where [Ca2+]i has been experimentally raised by depolarization from the resting level of 50–100 nM, it has been noted that a moderate increase to 150–250 mM in sympathetic neurons (Franklin et al. 1995) or to 200–400 nM in SGNs (Hegarty et al. 1997) results in survival; higher [Ca2+]i levels result in excitotoxicity (see Section 6 and Wangemann, Chapter 3). As further evidence of protection by elevated cytosolic Ca2+, increased levels of cytosolic Ca2+ (Tong et al. 1996) and increased levels of L-type voltage-gated Ca2+ channels (Koike and Tanaka 1991) have been implicated in the reduced dependence of mature neurons on peptide neurotrophic factors.

5.3.2 Ca2+ Signaling Pathways That Support SGN Survival

Ca2+ is an important second messenger (Berridge 1998); after Ca2+ entry via VGCCs, the increased level of cytosolic Ca2+ causes the activation of several Ca2+-dependent signaling systems. Most studies have focused on those in which the Ca2+-binding regulatory protein calmodulin (CaM) is involved. In SGNs, depolarization primarily promotes survival via at least three Ca2+/CaM-dependent signaling pathways: Ca2+/CaM-dependent protein kinase II (CaMKII), Ca2+/CaM-dependent protein kinase IV (CaMKIV), and protein kinase A (PKA) (Hansen et al. 2001b, 2003; Bok et al. 2003). These act independently and additively: activation of any one of these pathways promotes SGN survival, although not as effectively as depolarization (Hansen et al. 2001b, 2003) while, conversely, inhibition of any one of these pathways partially inhibits the ability of depolarization to support SGN survival (Hansen et al. 2001b, 2003; Bok et al. 2007). These Ca2+-dependent signaling pathways act in distinct subcellular compartments on distinct substrates, directed toward either cytoplasmic effectors or the cell nucleus. Thus, depolarization promotes SGN survival by inhibiting apoptotic signaling coordinately on the transcriptional and posttranslational levels.

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CaMKIV, which is primarily nuclear in SGNs, targets the transcription factor CREB and so constitutes a nuclear pathway—consistent with observations of other neurons (See et al. 2001). Genes regulated by CREB, such as Bcl-2 (Section 3.1), may therefore be important targets of depolarization-dependent signaling in SGNs.

CaMKII and CaMKII isoforms are expressed in SGNs (Bok et al. 2007). However, in contrast to CaMKIV, CaMKII promotion of survival is unaffected by CREB inhibition; possible nuclear targets for CaMKII include the kinesin family motor protein KIF4, which releases the prosurvival factor PARP-1 in a CaMKIIdependent manner to promote neuronal survival (Midorikawa et al. 2006). With regard to cytoplasmic CaMKII targets, CaMKII has been shown to activate the prosurvival transcriptional regulator NF- B in neurons (Meffert et al. 2003). Also, CaMKII in SGNs functionally inactivates the proapoptotic regulator Bad (Bok et al. 2007) and the proapoptotic protein kinase JNK (J. Huang and S.H. Green, unpublished observations).

The third Ca2+-dependent signaling pathway involves Ca2+/CaM-sensitive adenylyl cyclases that link depolarization to cAMP synthesis and activation of PKA. The second messenger cAMP is a prosurvival signal for neurons in general (Rydel and Greene 1988; Galli et al. 1995) and SGNs in particular (Hegarty et al. 1997). While cAMP, like CaMKs, appears to be a prosurvival signal in neurons, the mechanism may be different in different types of neurons. In CNS neurons, cAMP appears to promote survival by increasing responsiveness to neurotrophins (Meyer-Franke et al. 1998) but, in SGNs, cAMP promotes survival independently of neurotrophins (Hansen et al. 2001b). In SGNs (Bok et al. 2003) and other cells (Harada et al. 1999), the mechanism appears to involve phosphorylation and functional inactivation of the proapoptotic effector Bad by PKA, facilitated by the location of PKA action on the outer mitochondrial membrane (Harada et al. 1999; Affaitati 2003; Y.S. Cho, J. Huang, and S.H. Green, unpublished observations}. Although PKA can enter the nucleus and phosphorylate and activate CREB (De Cesare and Sassone-Corsi 2000), this appears to be irrelevant to support of SGN survival by cAMP. Rather, cytoplasmic PKA activity is necessary and sufficient for SGN survival promoted by cAMP signaling and nuclear activity is dispensable (Bok et al. 2003). Dominant-negative CREB mutants that blocked the ability of CaMKIV to promote survival had no effect on PKA (Bok et al. 2003).

Protein kinase C (PKC) is a CaM-independent Ca2+-activated protein kinase. PKC appears to be necessary, in part, for support of sympathetic neurons by NGF (Pierchala et al. 2004). Activation of PKC allows SGN survival in a MEKand PKB-dependent manner (Lallemend et al. 2003, 2005) but it is not known whether PKC activity is necessary for support of SGNs by depolarization. In contrast, PKC inhibition prevents programmed cell death in cerebellar Purkinje cells (Ghoumari et al. 2002), indicating that the consequences of particular Ca2+-dependent signals can be very different depending on the cellular context.

5.4 Suppression of JNK Signaling by Neurotrophic Stimuli

Jun phosphorylation and JNK activation can be detected in neurotrophic factor– deprived cultured SGNs and in SGNs in vivo after hair cell loss (Alam et al. 2007), implying a role in the death of deafferented SGNs. JNK inhibitors reduce death of neurotrophic factor–deprived cultured SGNs (Pirvola et al. 2000) and of SGNs in vivo after oxidative stress (Scarpidis et al. 2003). As noted in Section 4.1.1.4, MLKs are important upstream activators of JNK and MLK inhibitors prevent JNK activation after neurotrophic factor withdrawal. Peptide neurotrophic factors suppress JNK activation by preventing MLK activation. At least two distinct pathways are involved. Neurotrophic factor receptors inhibit the small GTPases Rac and Cdc42, which are MLK activators (Xu et al. 2001). Also, the protein-tyrosine kinase effector PKB can directly phosphorylate and inactivate MLKs (Barthwal et al. 2003).

Preliminary data on cultured SGNs indicates that, like peptide neurotrophic factors, depolarization suppresses JNK activation (J. Huang and S.H. Green, unpublished observations) and does so in a CaMKII-dependent manner.

5.5 Support of SGN Survival by Cells Other Than Hair Cells

The slow death of deafferented SGNs in the postnatal cochlea is strikingly different from the very rapid death of SGNs in NT-3−/− mouse embryos (Fritzsch et al. 2004). This may reflect the general loss of dependence on target-derived neurotrophic factors that occurs as neurons mature, which is due to multiple cellular changes. Another (not necessarily exclusive) explanation is that, in the mature cochlea, SGNs may receive survival-promoting stimuli from cells other than hair cells, so loss of the hair cells would not mean a complete lack of neurotrophic support, accounting for survival of some SGNs even long after hair cell loss. What could be the sources of such neurotrophic support? Neurons typically receive neurotrophic support from postsynaptic cells and it is likely that SGNs receive such support from the cochlear nucleus, because cutting the VIIIth nerve results in SGN death (Spoendlin 1971). It is also likely that SGNs can be supported by neurotrophic factors derived from paracrine/autocrine sources. Cell cultures, as well as freshly dissected spiral ganglia, contain BDNF and NT-3 (Hansen et al. 2001a, b; Zha et al. 2001). Survival of SGNs in cultures containing only cells from spiral ganglia (neurons and glia) is reduced when NT-3 or BDNF signaling is blocked (Hansen et al. 2001a, b). Last but not least, expression of NT-3 is not restricted to the inner hair cells, even in the mature cochlea, but is also present in supporting cells (inner pillar cell, inner phalangeal cell) (Sugawara et al. 2007), and long-term survival of SGNs after hair cell loss can be correlated with persistence of these supporting cells (Sugawara et al. 2005). Supporting cells in the mature cochlea play an active role in providing trophic support to SGN through the neuregulin-ErbB signaling pathway and genetic

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ablation of supporting cells therefore compromises SGN survival (Stankovic et al. 2004).

6. Excitotoxicity

Excitotoxicity is a neuronal trauma that can cause cell death or degeneration and may involve molecular mechanisms other than those participating in apoptosis due to loss of neurotrophic support (Mattson 2003). Excitotoxic death is principally due to an excessively high level of cytosolic Ca2+, generally the result of neural exposure to unusually high levels of excitatory neurotransmitter for prolonged periods (Mattson 2003). The principal excitatory neurotransmitter in the CNS (and in the inner ear) being glutamate, Ca2+ enters via Ca2+- permeable glutamate receptors—NMDA-type and some AMPA-type—and via voltage-gated Ca2+ channels that open as a result of the depolarization.

6.1 Excitotoxicity in the Cochlea

Excitotoxicity is seen in the cochlea mainly as a rapid destruction of the peripheral endings of type I SGNs after noise exposure. This is a result of the increased glutamate release from the inner hair cells, and these terminals can be effectively protected by intracochlear infusion of glutamate receptor antagonists (Puel et al. 1998). The SGNs themselves typically survive and the terminals regenerate. The ability of the SGNs to survive this insult is likely due to the fact that just a single postsynaptic site, well removed from the soma, is exposed to the glutamate and so that there may be only little Ca2+ entry in the soma. Consequently, it has been assumed that there is no long-term deleterious effect of noise-induced excitotoxicity on the SGNs themselves. However, recent work (Kujawa and Liebermann 2006) has shown that noise exposure in young mice causes an accelerated age-related hearing loss. This hearing loss is particularly unusual in that it is associated with a primary degeneration of the neurons rather than a primary loss of hair cells followed by secondary loss of neurons. Thus, while the neurons appear to recover from the noise, they may be compromised in a way that increases the probability of degeneration in the older mouse. Accelerated age-related hearing loss in individuals exposed to loud noise when young has also been observed in human epidemiological surveys (Gates 2006).

Excitotoxicity may be relevant to another inner ear pathology: Ménière’s disease involves the periodic exposure of the hair cells and spiral ganglion neurons to elevated extracellular K+ ([K+]) because rupture of the membranous labyrinth allows high K+ endolymph to contaminate the perilymph (Schuknecht 1993). High [K+] is directly toxic to hair cells (Zenner 1986) and to spiral ganglion neurons (Hegarty et al. 1997). In the latter case, the toxicity

is correlated with elevated cytosolic Ca2+ entering through voltage-gated Ca2+ channels (Hegarty et al. 1997), suggesting that this SGN death is excitotoxic. In extreme cases of Ménière’s disease, loss of hair cells or spiral ganglion neurons or both occurs, exacerbating the hearing loss (Schuknecht 1993).

6.2 Intracellular Mediators of Excitotoxicity

Excitotoxic cell death has some of the hallmarks of apoptosis, including proteolytic cleavage of key cellular proteins, but calpains—Ca2+-activated proteases—rather than caspases, appear to be the crucial proteases in excitotoxic death (Lankiewicz et al. 2000; Stefanis 2005) (although see Adamec et al. 1998). In agreement with this notion, genetic inhibition of caspases in transgenic mice in vivo by expression of the caspase inhibitor protein p35 does not prevent excitotoxic neuronal death (Higuchi et al. 2005). Analogous in vivo inhibition of calpains in transgenic mice by expression of the inhibitor protein calpastatin does inhibit excitotoxic neuronal death (Higuchi et al. 2005). In addition to calpain, the Ca2+-activated phosphatase calcineurin has been implicated in cell death acting by dephosphorylating and activating the proapoptotic effector Bad (Wang et al. 1999).

It should be noted that glutamate receptors and Ca2+ modulate other intracellular signals and some of these, notably nitric oxide (NO), appear to contribute to excitotoxicity (Araujo and Carvalho 2005). Also, NF- B deficiency increases the susceptibility of SGNs to excitotoxicity and accelerates age-related hearing loss due to SGN death (Lang et al. 2006).

6.3 LOC Efferents and Protection from Excitotoxicity

LOC efferents originate in the lateral superior olive and terminate on peripheral processes of SGNs. LOC efferents suppress auditory nerve activity in noisy conditions, presumably by inhibition at the postsynaptic bouton (Zheng et al. 1999; Groff and Liberman 2003). As discussed for the medial olivocochlear system in Section 4.1.1.6, this suppression of activity could also have a protective role; in this case, protection of SGNs. Three lines of evidence support such a role for the LOC. First, animals in which the entire olivocochlear projection is cut are much more vulnerable to acoustic injury than are animals in which only the crossed MOC projections were cut (Kujawa and Liberman 1997). Second, perfusing the dopamine (one of the neurotransmitters used by the LOC) receptor agonist piribedil into the cochlea protects against excitotoxic damage caused by noise or ischemia (d’Aldin et al. 1995). Third, dopamine in the LOC may be upregulated during sound conditioning (Niu and Canlon 2002). This would increase the efficacy of this system and contribute to the increased protection of preconditioning (Section 4.2).

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7. Therapeutic Interventions to Support SGN Survival After Loss of Hair Cells

Interest in SGN survival mechanisms derives in large part from the need to prevent SGN death in cochlear implant users. Death of all SGNs in the absence of hair cells would render the implant ineffective and make it impossible in the future to restore hearing via hair cell regeneration. Even if SGNs survive in the absence of hair cells, they lose their peripheral processes (Leake and Hradek 1988; Fayad et al. 1991; Nadol 1997). This presumably raises the threshold required to stimulate the neuron electrically, limiting the effectiveness and precision of stimulation by a cochlear implant. Strategies to support SGN survival in vivo in the absence of hair cells or to maintain or regrow the peripheral processes, are based on an understanding of SGN death and trophic support of SGNs in the normal cochlea. These are summarized in the text that follows and have also been reviewed elsewhere (Roehm and Hansen 2005).

While it might be expected that SGN survival plays a significant role in determining the efficacy of cochlear implants, the somewhat counterintuitive observation is that this does not appear to be the case. Rather, speech perception by cochlear implant users is not positively correlated with SGN number, provided that at least ≈10% of the neurons are present (e.g., Nadol and Eddington 2006). However, this may simply indicate that central rather than peripheral processing is the limiting factor for current technology: even a small number of SGNs is sufficient to convey to the CNS the limited amount of information provided by current cochlear implants and coding strategies. As the technology of cochlear implants improves, poor SGN survival or lack of the peripheral process may well become a limiting factor in their efficacy.

7.1 Protection of SGNs by Neurotrophic Factors In Vivo

Delivery of neurotrophic factors to rescue SGNs in vivo is a subject that has also been recently reviewed (Bianchi and Raz 2004; Roehm and Hansen 2005). BDNF, NT-3, and GDNF each will enhance SGN survival after hair cell loss. Two strategies have generally been used for delivery. In one, the factors are directly infused into the scala tympani via a microcannulation–osmotic pump system (Ernfors et al. 1996; Staecker et al. 1996; Miller et al. 1997; Keithley et al. 1998; Ylikoski et al. 1998). The second is a virally mediated gene therapy strategy in which a genetically modified virus is injected into the scala tympani, infecting cells lining the membranous labyrinth and causing them to produce and secrete neurotrophic factors (Staecker et al. 1998; Yagi et al. 2001; Kanzaki et al. 2002; Lalwani et al. 2002). These data support the efficacy of promoting SGN survival in vivo in the absence of hair cells by supplying peptide neurotrophic factors to which the neurons respond because they are likely to be exposed to them in vivo in the normal cochlea.

As noted in the preceding text, in vitro studies (Hartnick et al. 1996; Hegarty et al. 1997; Marzella et al. 1997, 1998) have shown that combining peptide

neurotrophic factors results in an approximate additive increase in SGN survival. Similar results have been obtained in vivo: BDNF combined with FGF-1 was shown to be more effective than either factor alone (Altschuler et al. 1999). Increased SGN survival was seen even if administration started after SGN degeneration had begun (Yamagata et al. 2004). Moreover, the enhanced SGN survival, induced by BDNF with CNTF, was associated with improved electrical excitability of the auditory nerve (Shinohara et al. 2002).

For translation and human application, there are a number of outstanding issues that require resolution, having to do with technical aspects of drug delivery and side effects of neurotrophic factors. For gene therapy, the primary issues relate to safety and efficacy of the vector as well as safety of the drug. Current data suggest that neurotrophic factor therapy, once initiated, must be maintained continuously. After cessation of BDNF infusion, SGNs rapidly degenerated so that, within 2 weeks after cessation, SGN density had fallen to the same level as without intervention (Gillespie et al. 2003). While direct drug infusion may be possible in conjunction with cochlear implantation, long-term presence of a cannula in the cochlea poses a significant risk of infection and is unlikely to be practical. Rather, gene therapy approaches based on those used in animal studies appear more promising.

7.2 SGN Survival in Response to Electrical Stimulation In Vivo

SGN death after loss of hair cells is significantly reduced if SGNs are stimulated by an implanted electrode (reviewed in Miller 2001). These results, obtained from studies of cats and guinea pigs, imply that deaf humans with cochlear implants should also experience reduced loss of SGNs.

SGN rescue may depend on place and intensity of the electrical stimulation (ES; Leake et al. 1991). The effect was greatest in the vicinity of the stimulating electrode. In kitten, lower intensity of ES (near-threshold for excitation) appears more effective than higher levels (Leake et al. 1995), although threshold sensitivity may have been changing throughout the course of the stimulation in these studies (Miller 2001). In the mature guinea pig, higher levels of ES appear most effective (Miller and Altschuler 1995; Mitchell et al. 1997). When stimulation was delayed for 2 or 4 weeks after deafening, in the guinea pig, SGN protection was reduced and the threshold for ES-induced protections was elevated (Miller and Altschuler 1995; Mitchell et al. 1997). The reduction in protection may be a consequence of a smaller surviving population of SGNs through the delay in initiating chronic ES.

Some studies of electrical stimulation in vivo did not find increased SGN survival after hair cell destruction in kitten (Araki et al. 1998, 2000) and in mature guinea pig (Li et al. 1999). Recently Shepherd and colleagues (Shepherd et al. 2005), while corroborating their earlier finding of no ES-induced SGN rescue, did report an ES-induced enhancement in SGN size, consistent with