Animal models of sensorineural hearing loss caused by exposure to noise and ototoxic agents have been established and well-characterized morphologically and functionally for several decades. In many of these models a rapid and robust loss of hair cells was seen before a significant loss of SGNs. However, in a study of aged rat ears, Keithley and Feldman (1982) reported that neuronal degeneration exceeded IHC loss, supporting the hypothesis that neuronal degeneration is not simply retrograde degeneration after loss of IHCs, but is an intrinsic degenerative process. Primary degeneration of SGNs was also seen in aged human cochlea without a robust loss of sensory hair cells (Schuknecht & Gacek, 1993; Makary et al., 2011). In addition, primary neural degeneration was reported in some cases of noise trauma (Spoendlin, 1971; Liberman & Mulroy, 1982), aminoglycoside ototoxicity (Sone et al., 1998), and in the cochleas of white cats with hereditary deafness (Pujol et al., 1977), suggesting SGN degeneration is not a unique secondary event. A series of previous studies have found that degeneration of afferent synapses and progressive loss of SGNs occur in the cochlea when the sensory hair cells are still intact and functional after exposure to an octave-band noise at moderate levels (Kujawa & Liberman, 2006, 2009; Lin et al., 2011; see Fig. 8.4). These data strongly support that SGN degeneration can be independent of the loss of sensory hair cells.

Glutamate is the most common excitatory neurotransmitter in the central nervous system and is believed to play an important role in cochlear mechano-neural transduction (Bird et al., 1978; Fuchs et al., 2003). The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor has been identified in type I SGNs (Pujol et al., 1985; Liberman et al., 2011). The excessive release of glutamate results in neuronal damage through excitotoxicity. The pathophysiology of excitotoxicity includes overactivation of glutamate receptors, influx of high levels of calcium ions (Ca²⁺) to the postsynaptic cells, and neuronal cell death (Hutchins & Barger, 1998; Martin et al., 1998).

It has been hypothesized that primary SGN degeneration occurs by means of excitotoxic neural damage. Pathological characteristics of primary SGN degeneration include massive swelling of afferent nerve terminals under the basal pole of IHCs and a total disruption of the postsynaptic membrane (Fig. 8.3; Robertson, 1983; Puel et al., 1998). The pathological alterations of afferent dendrites may be caused by excessive presynaptic release of the neurotransmitter glutamate after acoustic stimulation (Eybalin, 1993; Puel et al., 1998; Hakuba et al., 2000). Local application of glutamate agonists can induce pathologic changes in afferent dendrites similar to those induced by noise trauma (Pujol et al., 1985; Zheng et al., 1997) and the glutamate antagonist kynurenate can protect SGN dendrites from acoustic damage (Puel et al., 1998). Afferent terminals can fully or partially recover from excitotoxic damage and this recovery may play a role in the phenomenon of temporary threshold shift (TTS) (Liberman & Mulroy, 1982; Robertson, 1983; Puel et al., 1996). However, loss of presysnaptic ribbons and progressive SGN loss after TTS has also been reported in recent studies (Kujawa & Liberman, 2006, 2009; Fig. 8.4).

Primary degeneration of SGNs after noise exposure has been understood largely based on experiments associated with glutamate receptor antagonists. However, questions remain on several critical issues. First, high-level noise exposures (e.g., 130 dB SPL pure tone used by Puel et al., 1998) were often applied to generate at least two types of cochlear lesions: (1) loss of hair cells starting with OHCs at lower levels then including IHCs at higher levels; and (2) a massive destruction of afferent terminals below IHCs as shown in Fig. 8.3. Although pathological nerve alterations were seen as early as 20 min after sound exposure (suggesting this pathology is independent of hair cell loss), direct evidence of primary degeneration is still needed from a model with only auditory nerve injury. Also, an evaluation of whether excitotoxic lesions in the afferent nerve terminals are able to recover fully and whether auditory nerves regenerate after noise trauma is still needed. Until recently, most of the morphological observations in these studies were performed at the ultrastructural level. Longitudinal evaluations of dynamic changes in afferent synapses and afferent nerve terminals under IHCs are extremely challenging, and quantitative analysis of morphological alterations in the auditory nerve is virtually absent from these earlier studies. Finally, because lesions are mixed in these models, comprising losses of afferent synapses, SGNs, IHCs, and OHCs, it is difficult to determine which component contributes to the various noise-induced auditory functional deficits.

Recently, newly developed genetic, biochemical, electrophysiological, and high-resolution optical approaches have provided tools for the quantitative examination of the degeneration of SGNs and their associated elements (Khimich et al., 2005; Weisz et al., 2014; Rutherford and Moser, Chap. 5). Numerous biological markers for synapses and nerve terminals have been identified and characterized, including antibodies for the presynaptic ribbon (RIBEYE/transcription factor CtBP2; Khimich et al., 2005), postsynaptic glutamate receptor patches (GluR2/3; Matsubara et al., 1996), unmyelinated nerve terminals (neurofilament; Berglund & Ryugo, 1991), and afferent terminal swellings (parvalbumin; Kujawa & Liberman, 2009). A series of studies using (1) high-powered confocal imaging of sensory epithelium, (2) three-dimensional quantification of ribbon synapse numbers, and (3) histological quantification of the neuronal cells demonstrated that a moderate level of noise exposure can cause a permanent loss of afferent synapses without hair cell damage (Kujawa & Liberman, 2009; Lin et al., 2011; Furman et al., 2013). These studies showed a rapid and selective loss of afferent synaptic ribbons under IHCs after progressive degeneration of SGNs occurring in mice after exposure to an 8–16-kHz octave-band noise at 100 dB SPL for 2 h (Fig. 8.4). Shortly after noise exposure, auditory brain stem responses (ABRs) were elevated about 40 dB concurrent with a slightly smaller threshold elevation of distortion product otoacoustic emissions (DPOAEs)—a measure of OHC function. By 2 weeks after noise exposure, ABR and DPOAE thresholds were back to normal, preexposure levels. Even though cochlear threshold sensitivity fully recovered, the ABR wave I amplitudes were reduced significantly at high stimulus levels at frequencies strongly affected by the noise. Together, these groundbreaking studies provide direct evidence that primary degeneration can occur in the inner ear in response to pathological stress conditions.

Genetic studies of sensorineural hearing loss have progressed at a rapid pace in recent years. To date, more than 64 genes and 125 loci that link to various degrees of hearing impairment have been identified (reviews by Dror & Avraham, 2010; Angeli et al., 2012). Some of these genes play important roles in the regulation of synaptic transmission and neuronal survival and death. Deficiency of these genes likely contributes to primary SGN degeneration. Santarelli (2010) reviewed the genes associated with human auditory neuropathy, diagnostically characterized as having abnormal ABRs and completely preserved otoacoustic emissions (OAEs). Here, a brief review was given on several well-documented genes that are associated with auditory neuropathy—SLC17A8, OTOF, PJVK, and DIAPH3. In addition, animal studies of gene defects revealed two transcription factors, nuclear factor κB (NF-κB) and forkhead box O3 (FoxO3), that play important roles in maintaining the survival of SGNs and normal function of the auditory nerve and the IHC synapse (Lang et al., 2006; Gilels et al., 2013).

Vesicular glutamate transporter VGLUT3 (SLC17A8, DFNA25) and otoferlin (OTOF, DFNB9) are two key components of the afferent synapse on IHCs. VGLUT3, one of the three subtypes of vesicular glutamate transporters (VGLUT 1-3), is selectively expressed in IHCs and responsible for loading the synaptic vesicles with glutamate (Ruel et al., 2008; Seal et al., 2008). Mice lacking VGLUT3 have no auditory brainstem responses but have robust OAEs, indicating an appearance of normal OHC function. A significant reduction of IHC synapse numbers and pathological alterations of SGNs were also observed in these mice. Otoferlin is a multi-C₂ domain protein essential to the exocytosis of synaptic vesicles in IHCs and the consequent action of the Ca²⁺ sensor triggering membrane fusion at the IHC ribbon synapse (Yasunaga et al., 1999; Roux et al., 2006). Otoferlin-deficient mice (Otof ^−/−) lacking exons 14 and 15, which encode most of C₂C domain, are totally deaf but have preserved OAEs. Although normal IHC ribbon synapses were observed in postnatal Otof ^−/− mice, the pathological alterations of SGNs have not been determined (Roux et al., 2006).

Pejvakin, encoded by PJVK, is a 352-residue protein belonging to the gasdermin protein family and is expressed in cochlear hair cells, supporting cells, and SGNs (Delmaghani et al., 2006). Abnormal expression of this protein is associated with nonsyndromic auditory neuropathy DFN59 and also DFNA5, which participates in the p53-regulated cellular response to DNA damage (Masuda et al., 2006). Mice lacking pejvakin (Dfnb59t ^m1Ugds ) show an elevation of ABR thresholds but normal OAEs at affected frequencies. Examination of the organ of Corti via scanning electron microscope revealed no structural abnormalities, but a detailed examination of auditory nerve morphology was not included. The Diaphanous homolog 3 (DIAPH3), which encodes the diaphanous-3 protein, was mapped to the autosomal dominant auditory neuropathy, dominant 1 (AUNA1). Analysis of lymphoblastoid cells showed an upregulation of DIAPH3 mRNA expression suggesting a gain of function effect present in AUNA1-affected patients (Schoen et al., 2010). Expression of a constitutively active form of the diaphanous protein in Drosophila leads to a deficiency of auditory response from the auditory organ.

The transcription factor NF-κB has a fundamental role in regulating inflammatory responses and apoptosis in response to injury in many cell types (Barkett & Gilmore, 1999). The p50/p65 heterodimer is the predominant complex of NF κB in most mammalian cells. NF-κB is expressed and shows a low-level constitutive activity in the neurons of the central nervous system (Kaltschmidt et al., 1994). By using measures of cochlear function and histopathological evaluation, an accelerated hearing loss with correlated primary degeneration of SGNs and afferent nerve processes was seen in the SGNs of p50^−/− mice (Lang et al., 2006). As shown in Fig. 8.5, marked excitotoxic-like alterations were seen at afferent terminals under IHCs of young adult p50^−/− mice (1–3 month old). In contrast, no major pathological changes were seen in OHCs or the stria vascularis in the same cochleas. At 8 months of age, the density of SGNs in the basal turn of the knockouts was only about half that of wild-type mice. However, neither significant EP loss with age nor accelerated degeneration of hair cells was seen in the same cochleas, indicating that the loss of SGNs and auditory nerves is primary and independent of the degeneration of sensory hair cells.

FoxO3 is a transcription factor belonging to the forkhead O subclass, which is characterized by a distinct forkhead DNA-binding domain. It plays an important role in the regulation of stress response proteins in a variety of pathological conditions, including excitotoxic damage in brain tissue (Brunet et al., 1999; Davila et al., 2012). Adult mice lacking FoxO3 have elevated ABR thresholds but normal OHC function. Comprehensive histological examinations of cochlear tissues revealed that alterations of synapse locations and degeneration of the afferent nerve cause age-related hearing loss in these mice (Gilels et al., 2013).