Deafferentiation-Associated Changes in Afferent and Efferent Processes in the Guinea Pig Cochlea and Afferent Regeneration With Chronic Intrascalar Brain-Derived Neurotrophic Factor and Acidic Fibroblast Growth Factor

Deafferentation of the auditory nerve from loss of sensory cells is associated with degeneration of nerve fibers and spiral ganglion neurons (SGN). SGN survival following deafferentation can be enhanced by application of neurotrophic factors (NTF), and NTF can induce the regrowth of SGN peripheral processes. Cochlear prostheses could provide targets for regrowth of afferent peripheral processes, enhancing neural integration of the implant, decreasing stimulation thresholds, and increasing specificity of stimulation. The present study analyzed distribution of afferent and efferent nerve fibers following deafness in guinea pigs using specific markers (parvalbumin for afferents, synaptophysin for efferent fibers) and the effect of brain derived neurotrophic factor (BDNF) in combination with acidic fibroblast growth factor (aFGF). Immediate treatment following deafness was compared with 3-weekdelayed NTF treatment. Histology of the cochlea with immunohistochemical techniques allowed quantitative analysis of neuron and axonal changes. Effects of NTF were assessed at the light and electron microscopic levels. Chronic BDNF/aFGF resulted in a significantly increased number of afferent peripheral processes in both immediateand delayed-treatment groups. Outgrowth of afferent nerve fibers into the scala tympani were observed, and SGN densities were found to be higher than in normal hearing animals. These new SGN might have developed from endogenous progenitor/stem cells, recently reported in human and mouse cochlea, under these experimental conditions of deafferentation-induced stress and NTF treatment. NTF treatment provided no enhanced maintenance of efferent fibers, although some synaptophysin-positive fibers were detected at atypical sites, suggesting some sprouting of efferent fibers. J. Comp. Neurol. 507:1602–1621, 2008. © 2008 Wiley-Liss, Inc. Indexing terms: cochlea; nerve fiber regeneration; brain-derived neurotrophic factor; acidic fibroblast growth factor Grant sponsor: European Community; Grant number: QLG3-CT-200201463; Grant sponsor: National Institutes of Health; Grant number: NIHNIDCD R01 DC003820; Grant number: P30 DC005188; Grant sponsor: Austrian Science Foundation; Grant number: P15948-B05; Grant sponsor: General Motors Corporation; Grant sponsor: Ruth and Lynn Townsend Professorship of Communication Disorders. *Correspondence to: Prof. Anneliese Schrott-Fischer, Department of Otolaryngology, Medical University of Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria. E-mail: [email protected] Received 30 April 2007; Revised 17 July 2007; Accepted 3 December 2007 DOI 10.1002/cne.21619 Published online in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 507:1602–1621 (2008) © 2008 WILEY-LISS, INC. Deafferentation of the auditory nerve from loss of sensory cells (inner hair cells; IHC) is associated with the loss of auditory function. As IHC are damaged, they release excess glutamate, resulting in the swelling and bursting of the peripheral processes of the auditory nerve that had been connected to the IHC. These auditory nerve processes are capable of recovery and reconnecting if IHC recover (Puel et al., 1995, 1998); in the presence of massive IHC loss, however, these processes do not recover. The unmyelinated portion of the auditory nerve peripheral processes rapidly regresses to the habenula perforata, followed by a slower regression of myelinated portion toward the cell bodies of the auditory nerve [spiral ganglion neurons (SGN); Webster and Webster, 1981; Koitchev et al., 1982; Bichler et al., 1983; Spoendlin, 1984; Morest et al., 1998]. As with deafferentation-associated cell death in other systems, SGN apoptosis appears to be induced by the loss of survival factors, including neurotrophic factors, following IHC destruction (for reviews see Miller et al., 2002; Roehm and Hansen, 2005). Apoptosis of SGN (Roehm and Hansen, 2005) proceeds gradually over a period of time, with the rate of cell death occurring more rapidly in rat, mouse, chinchilla, or guinea pig (Webster and Webster, 1981; Koitchev et al., 1982; Bichler et al., 1983; Jyung et al., 1989; McFadden et al., 2004), compared with cat (Leake and Hradek, 1988) and primate, including man (Suzuka and Schuknecht, 1988; Nadol et al., 1989; Nadol, 1990, 1997; Zimmermann et al., 1995; Incesulu and Nadol, 1998). In the guinea pig, there is a large SGN loss by 2 months following hair cell loss (Webster and Webster, 1981; Jyung et al., 1989). The loss of SGN can have serious clinical repercussions. Cochlear prostheses are now used to restore hearing successfully following deafness from sensory cell loss in large numbers of patients. Cochlear prostheses, however, depend on direct electrical stimulation of the auditory nerve neurons for their function, and loss of SGN can compromise their efficacy. Studies have now shown that SGN survival following sensory cell loss can be significantly enhanced by application of neurotrophic factors (NTF), such as brain-derived neurotrophic factor (BDNF; Staecker et al., 1996; Miller et al., 1997; Gillespie et al., 2003; Shepherd et al., 2005) neurotrophin-3 (NT-3; Ernfors et al., 1996), glial cell line-derived neurotrophic factor (GDNF; Ylikoski et al., 1998; Altschuler et al., 1999; Yagi et al., 2000), and combinations of neurotrophic factors such as BDNF and fibroblast growth factor (FGF; Altschuler et al., 1999) or BDNF and ciliary neurotrophic factor (CNTF; Yamagata et al., 2004). NFT are also involved in the formation and maintenance of afferent and efferent cochlear connections (Fritzsch et al., 2004) during normal development. The same NFT that enhance SGN survival might then also induce the regrowth of their peripheral processes, even in the absence of IHC (Ernfors et al., 1996; Staecker et al., 1996; Altschuler et al., 1999; Wise et al., 2005). Regrowth of peripheral processes could enhance the integration of implant and auditory nerve, increase electrode-nerve process proximity, lower excitatory thresholds, and increase selectivity of activation. This could, in turn, improve efficacy and function of cochlear prostheses. Previous studies, however, have used nerve fiber markers that could not differentiate whether the regrowth was from afferent processes of the auditory nerve, from lateral and medial efferent processes to the cochlea, or from some combination of afferents and efferents. Regrowth of lateral or medial efferents would have unknown benefit to the efficacy of cochlear prostheses. This study is based on the hypothesis that neurotrophic treatment selectively enhances afferent fiber regrowth and uses selective markers that differentiate afferent and efferent processes in the cochlea. Parvalbumin is a calcium-buffering protein that in the cochlea selectively labels SGN and their processes as well as IHC (Eybalin and Ripoll, 1990; Soto-Prior et al., 1995; Pack and Slepecky, 1995). Synaptophysin is a synaptic vesicleassociated protein that provides specific labeling of olivocochlear efferent fibers and terminals in the cochlea (GilLoyzaga and Pujol, 1988; Nadol et al., 1993; Knipper et al., 1995; Simmons et al., 1996; Counter et al., 1997). The present study used parvalbumin as a marker for the peripheral processes of the auditory nerve and synaptophysin as a marker for olivocochlear efferent peripheral processes in immunocytochemical investigation of the progression of deafness-associated changes in efferent and afferent peripheral processes and the influence of chronic intrascalar administration of BDNF and aFGF on regrowth and maintenance of afferent vs. efferent processes. MATERIALS AND METHODS Study groups and design Studies were performed in pigmented 250–350-g guinea pigs of both genders from Elm Hill Breeding Labs (Chelmsford, MA). Animals were housed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, with free access to food and water throughout the duration of the experiment. Veterinary care and animal husbandry were provided by the Unit for Laboratory Animal Medicine at the University of Michigan, and all protocols were approved by the University Committee for the Use and Care of Animals at the University of Michigan. Experiments were performed in accordance with the guidelines of the National Institutes of Health Guide for the care and use of laboratory animals (NIH Publications No. 80-23, revised 1978). A concerted effort was made to minimize both the number of animals used and the suffering of subjects involved in the study. Normal hearing by baseline acoustic auditory brainstem response (aABR 1) recording was necessary for inclusion in the study. Animals were then randomly divided into eight groups (Fig. 1). Group 1 animals were nontreated, normal hearing controls. Groups 2–8 were systemically deafened with kanamycin (450 mg/kg) SQ followed (2 hours later) with ethacrynic acid (60 mg/kg). Deafness was confirmed by acoustic auditory brainstem response (aABR 2) to a click stimulus (Miller et al., 2007) with a 60-dB SPL threshold shift necessary for continued inclusion (groups 2 and 4 were tested 3 days following deafening; groups 3 and 5–8 were tested 5–7 days following deafening). Groups 2, 3, and 6 received no further treatment after they were deafened and were assessed 3, 7, and 21 days following deafening, respectively. Two groups (groups 4 and 7) received NTF treatment. Group 4 received 26 days of chronic intrascalar infusion of BDNF (100 g/ml) and aFGF (50 ng/ml) in one ear from a miniosmotic pump with a cannula into scala tympani (as in Brown et al., 1993; Prieskorn and Miller, 2000), with treatment starting 3 days following deafening (PD3 The Journal of Comparative Neurology. DOI 10.1002/cne 1603 AFFERENT REGENERATION WITH INTRASCALAR BDNF AND AFGF