www.elsevier.com/locate/ynbdi Neurobiology of Disease 18 (2005) 184 – 192 NGF stimulates extensive neurite outgrowth from implanted dorsal root ganglion neurons following transplantation into the adult rat $ inner ear Zhengqing Hu,a,b,* Mats Ulfendahl,a,b,* and N. Petri Oliviusa,b,c a Center for Hearing and Communication Research, Karolinska Institute, SE-171 76 Stockholm, Sweden Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden c Department of Otorhinolaryngology, Karolinska Hospital, Stockholm, Sweden b Received 30 March 2004; revised 31 August 2004; accepted 13 September 2004 Available online 10 December 2004 Neuronal tissue transplantation is a potential way to replace degenerated spiral ganglion neurons (SGNs) since these cells cannot regenerate in adult mammals. To investigate whether nerve growth factor (NGF) can stimulate neurite outgrowth from implanted neurons, mouse embryonic dorsal root ganglion (DRG) cells expressing enhanced green fluorescent protein (EGFP) were transplanted into the scala tympani of adult rats with a supplement of NGF or artificial perilymph. DRG neurons were observed in the cochlea for up to 6 weeks postoperatively. A significant difference was identified in the number of DRG neurons between the NGF and non-NGF groups. In the NGF group, extensive neurite projections from DRGs were found penetrating the osseous modiolus towards the spiral ganglion. These results suggest the possibility that embryonic neuronal implants may become integrated within the adult auditory nervous system. In combination with a cochlear prosthesis, a neuronal implantation strategy may provide a possibility for further treatment of profoundly deaf patients. D 2004 Elsevier Inc. All rights reserved. Keywords: Cochlear implant; Dorsal root ganglion; Hair cell; Hearing loss; Nerve growth factor; Neurite; Spiral ganglion; Transplantation Introduction It has been widely accepted that the auditory system in mammals cannot regenerate spontaneously following insults to the cochlear sensory epithelium (hair cells), that is, following acoustic overstimulation, ototoxic drugs, or genetic disorders $ Neurite outgrowth of DRG following implantation into cochlea. * Corresponding authors. Center for Hearing and Communication Research, Building M1, Karolinska University Hospital, SE-171 76 Stockholm, Sweden. Fax: +46 8 301876. E-mail addresses: Zhengqing.hu@cfh.ki.se (Z. Hu), Mats.ulfendahl@cfh.ki.se (M. Ulfendahl). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.09.010 (Holley, 2002; Rubel and Fritzsch, 2002). Moreover, in the auditory system, as in other afferent nervous systems, degeneration of the spiral ganglion neurons (SGNs) and peripheral dendrites occurs secondarily to the loss of the cochlear sensory epithelium, thereby aggravating the impairment of the auditory system (Ernfors et al., 1995; Marzella and Clark, 1999; Nadol et al., 1989; Ryals et al., 1989). A cochlear implant is presently the most effective therapy for patients suffering from profound sensorineural hearing loss (Gantz et al., 2002; Gomaa et al., 2003; Shepherd and Hardie, 2001). The implanted electrode can directly stimulate the SGNs or their peripheral processes, thereby bypassing the damaged cochlear sensory epithelium. Observations from animal experiments (Pfingst et al., 1981) as well as from human clinical and histological findings (Hardie and Shepherd, 1999; Leake and Hradek, 1988; Nadol, 1997; Nadol and Xu, 1992) have illustrated that the postoperative hearing ability is dependent not only on the efficacy of the cochlear implant device, but also on the functional integration of SGNs. Based on a limited number of histological observations, there has been a debate about the relationship between the number of SGNs and the postoperative implant performance (Fayad et al., 1991; Nadol et al., 2001). It is believed that the degeneration of SGNs will reduce the efficiency of the cochlear nerve to response to electrical stimulation (Hardie and Shepherd, 1999; Shepherd and Javel, 1997; Zhou et al., 1995) and that a minimum number of functional SGN is required to achieve a high level of perception in cochlear implant patients (Blamey, 1997). A recent study demonstrates that neurotrophins can increase SGNs survival in the deafened animals which consequently results in a better threshold of the electrical acoustic brainstem response (Shinohara et al., 2002). However, profound sensorineural hearing loss patients will ultimately have an extensive loss of the number and function of SGNs. As a consequence, a cochlear prosthesis may not have the minimum number of functional SGNs to stimulate, leading to a suboptimal function of the device. One way to restore degenerated or missing SGNs may be cell replacement therapy (Hu et al., 2004b; Olivius et al., 2003, 2004). 185 Z. Hu et al. / Neurobiology of Disease 18 (2005) 184–192 growth cone migration and axonal branching (Gallo and Letourneau, 1998; Gallo et al., 1997), and promote regeneration of axons (Bloch et al., 2001; Edstrom et al., 1996; Terenghi, 1999). In order to (1) enhance the survival of the implanted DRG neurons, (2) stimulate the neurite outgrowth of transplanted neurons, and (3) establish possible connections between the DRG neurons and the host SGNs, in this study, NGF was infused into the inner ear together with the implantation of DRG neurons. Materials and methods Fig. 1. Transplantation of mouse embryonic DRGs into the adult inner ear. The DRGs were transplanted into the scala tympani of adult rat. The spiral ganglion neurons are located within the bony modiolus (Rosenthal canal). The challenges for a successful inner ear cell replacement strategy include not only the survival and migration of the implanted neurons to the functionally relevant area (e.g., spiral ganglion region, c.f., Fig. 1), but also the integration of the implanted neurons within the host auditory system, thereby creating the possibility for the establishment of a functional unit following the transplantation. The selection of an appropriate cell candidate is a key issue. Dorsal root ganglion (DRG) neurons may not be the only feasible alternative for replacing SGNs. However, the implantation of DRGs may provide relevant information regarding the implantation of various tissues, that is, SGNs and stem cells into the inner ear (Hu et al., 2004c). Furthermore, our previous experiments have shown that embryonic DRG neurons cannot only survive in the adult inner ear (Hu et al., 2004b; Olivius et al., 2003, 2004), but also migrate towards Rosenthal canal and within the spiral ganglion (Hu et al., 2004b). These studies, however, did not focus on any potential connections between the transplanted neurons and the host SGNs. It is well documented that neurotrophic factors [e.g., nerve growth factor (NGF)] possess the ability to regulate neuronal survival (Crowley et al., 1994; Minichiello et al., 1995; Zimmermann, 1998), are involved in the retrograde interactions between neurons and their targets (DiStefano et al., 1992; Snider and Wright, 1996), locally stimulate All animal procedures were approved by the regional ethical committee (approval 283a-d/02 and 464/03). DRG implantation was made into a total of 24 Sprague–Dawley rats (body weight 250–280 g). The host animals were paired by gender and body weight, and equally divided into two experimental groups: animals supplied with NGF into the inner ear (NGF group, n = 12) and animals supplied with artificial perilymph (non-NGF group, n = 12). These two groups were further divided into a 3-week-survival group (n = 6) and a 6-weeksurvival group (n = 6, c.f., Table 1). Preparation of DRGs The dissection of DRGs has been described previously (Hu et al., 2004b; Olivius et al., 2003, 2004). Briefly, donor DRG neurons were dissected out from mouse embryos at embryonic days 13–14 (E13–14). The animals were from a transgenic mouse line with an enhanced green fluorescent protein (EGFP) cDNA under the control of a chicken h-actin promoter and cytomegalovirus enhancer [strain C57BL/6-TgN (ACTbEGFP) 1Osb from The Jackson Laboratory, Bar Harbor, Maine]. Under aseptic conditions and deep anesthesia (ketamine, Pfizer AB, T7by, Sweden, 4 mg/100 g; and xylazine, Bayer AG, Leverkusen, Germany, 1 mg/100 g; i.m.), the abdomen and uterus of the pregnant mice were exposed. The embryos were excised, decapitated, and transferred to tissue culture medium (DMEM, Gibco BRL Life Technologies, Eggenstein, Germany). Two lower lumbar DRGs were identified and dissected out on each side of the spinal cord. The DRGs were then transferred to the culture medium and kept at 48C until transplantation. The Table 1 The survival and outgrowth patterns of DRG neurons following implantation into the cochlea Experimental group NGF group Survival period Number of animals Per group With DRG implant survival With implants close to SG and OC Showing neurite projections to SGNs Number of surviving DRG neurons Length of DRGs neurite outgrowth (Am) Diameter of surviving DRG neurons (Am) 3 weeks 6 weeks 3 weeks Non-NGF group 6 weeks – t Test 6 6/6 6/6 3/6 332 F 36 55.9 F 22.2 18.0 F 2.7 6 2/6 2/6 0/6 – – – 6 4/6 4/6 0/6 135 F 14 14.8 F 7.4 17.6 F 2.9 6 0/6 0/6 0/6 – – – – – – – P b 0.01 P b 0.01 P N 0.05 Implanted mouse embryonic DRG neurons show higher survival rates and neurite outgrowth towards the adult rat auditory system. The implanted DRG neurons are found close to spiral ganglion (SG) and the organ of Corti (OC). Neurite outgrowth (the axons positively labeled by the neurofilament antibodies) was observed between the transplanted DRG neurons and the host spiral ganglion neurons (SGNs) in the NGF group at 3 weeks following implantation. Analysis (two-tailed, paired t test) shows that there is a statistically significant difference in the number of surviving DRG neurons between the NGF group and the non-NGF group at a 3-week postoperative survival. 186 Z. Hu et al. / Neurobiology of Disease 18 (2005) 184–192 pregnant mice were euthanized by an overdose of pentobarbital (i.p.) postoperatively. To analyze the morphology and number of the nontransplanted DRG neurons, a total of 12 lower lumbar DRGs were dissected out from three embryos. For control purpose, six out of these DRGs were randomly selected and fixed in 4% paraformaldehyde (10 min), then cryosectioned (12 Am) and analyzed. EGFP detection All the sections from each rat cochlea were collected and observed using a Zeiss fluorescence microscope with a digital camera (Spot RT Diagnostic Instrument, Optical Elements Corp., Dulles, VA) or a confocal microscope (Zeiss LSM 510, Oberkochen, Germany). Immunohistochemistry Transplantation of DRGs into the inner ear The surgical procedure for transplanting DRGs has been described before (Hu et al., 2004b; Olivius et al., 2003, 2004). Briefly, under deep anesthesia (same drugs and dosages as mouse, body weight-adjusted), the left postauricular area of the rat was shaved and rinsed with 70% ethanol, and the animal was placed on a heating pad with the temperature set at 378C to maintain body temperature during surgery. Under aseptic conditions, the left bulla was exposed and opened to provide access to the cochlea with a postauricular approach. The basal cochlear turn was identified, and a small hole was made into the scala tympani using a fine diamond drill. Four lumbar DRGs from the same mouse embryo were transplanted into two host rats which were paired by gender and body weight; two DRGs were transplanted into the NGF group rat, and the other two DRGs were implanted into the non-NGF group animal. A fine catheter was inserted into the scala tympani and connected to a mini-osmotic pump (Alzet Model 2002, Alza Corp., Palo Alto, CA; pump capacity 0.5 Al/h, duration 2 weeks). A small piece of fascia was placed over the cochleostomy; the catheter was fixed with dental cement on the surface of the bulla, the pump was kept subcutaneously on the back of the animal, and the skin incision was approximated with sutures. The mini-osmotic pump was set to pump either NGF (120 ng/h; Sigma–Aldrich, Sweden AB, Stockholm, Sweden; 240 Ag/ ml, diluted in artificial perilymph) or as a control the same volume (250 Al) of artificial perilymph as the non-NGF group. The artificial perilymph had the following composition (in mM): NaCl, 137; KCl, 2.8; CaCl2, 1.5; MgCl2, 1.0; NaH2PO4, 8.0; KH2PO4, 4.7; glucose, 11.0; pH 7.4 (Olivius et al., 2003; Shah et al., 1995). The duration of the pump was 14 days. The artificial perilymph was filtered with a 0.22-Am filter before NGF was added into it. The preparation of all the solutions for use in the mini-osmotic pumps was done in an aseptic condition. In order to reduce the risk of postoperative immunological rejection or infection, the transplanted host animals received daily injections of cyclosporin (Novartis Sverige AB, T7by, Sweden; 0.56 mg/100 g body weight) and doxycycline (Nordic Drugs AB, Malmf, Sweden; 0.24 mg/100 g body weight) intraperitoneally until the day of euthanization. Histology The animals were euthanized with an overdose of pentobarbital. Once the pedal withdrawal reflex had been lost, the animals were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 48C following 0.9% saline at 378C. The cochleas were excised, trimmed, and kept in the fixative before being transferred to a 0.1 M phosphate buffer, in which they were stored shortly until further processing. The cochleas were further carefully trimmed under a dissecting microscope, decalcified in 0.1 M EDTA for approximately 1 week, and sectioned on a cryostat at a section thickness of 12 Am. A neurofilament antibody was used for immunohistochemical detection of the implanted mouse neuronal tissue (as compared to nonneural tissue which may accompany the implants) in sections with surviving DRG implants from the host animals. Briefly, following preincubation in goat serum (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), the sections were incubated with neurofilament antibody (1:100, commercially available at Santa Cruz Biotechnology, Inc.) overnight at 48C. After rinsing, the secondary antibody goat anti-mouse IgG1-Texas red (1:200, Santa Cruz Biotechnology, Inc.) was applied to the sections at room temperature for 4 h. In order to identify the subtype of the surviving DRG cells in the cochlea, substance P antibody [SP, for the detection of small size (b25 Am) DRG cells] and calcitonin gene-related peptide antibody [CGRP, for detection of small– medium size (25–40 Am) DRG cells] were used in the study. Using the same immunostaining protocol as above, primary antibodies (SP and CGRP) and secondary antibody (rabbit anti-goat IgGconjugated with Texas red) were applied on the histological sections of the cochlea (all the antibodies and serum were from Santa Cruz Biotechnology, Inc). The sections were observed using the same fluorescence microscope equipment as above with a Texas red fluorescence filter. DRG neuronal counts and statistical analyses DRG cell counts were performed on the sections from the 3week-survival animals (the NGF group and the non-NGF group host rats) with surviving DRG implants. Six nontransplanted DRGs from three embryos served as control. The number of surviving DRG neurons was counted and measured in every two sections using stereological techniques (Eriksson et al., 1994; Gundersen et al., 1988; Olivius et al., 2003, 2004). Only the neurons double-labeled with EGFP and neurofilament antibodies as well as showing clearly visible neuronal profiles containing one nucleolus were counted (c.f., Fig. 2). The diameter and the length of the neurite outgrowth (Am) of the surviving DRG neurons were measured using a SigmaScan image measurement system and a SigmaScan Pro automated image analysis software (Jandel Scientific Software, Erkrath, Germany). The diameter was calculated as the mean from the perpendicular longest and the shortest diameters for each neuronal periphery out of which the average diameters in the NGF and the non-NGF groups were calculated. In order to evaluate the length of the neurite outgrowth from the implanted neurons, 100 randomly selected axons from each group were measured. Since the DRGs were simultaneously transplanted into the gender- and body weightpaired animals of the NGF and the non-NGF groups, the number of neurons transplanted into each group was not regarded as different from the other group. The average [mean and standard deviation (SD)] number, diameter, and length of the neurite outgrowth of the surviving DRG neurons were computed in the NGF- and the non-NGF groups at 3 weeks following implanta- Z. Hu et al. / Neurobiology of Disease 18 (2005) 184–192 187 Fig. 2. Mouse DRG implants were found in the scala tympani at 3 weeks following transplantation into the adult rat cochlea. The implant was supported by exogenous NGF. A–C are from the histological section from one animal and D–E are from a different preparation. The EGFP fluorescence of the DRG neurons was readily identifiable (A, D). The DRG neurons were double-labeled with neurofilament antibodies (B, E) and merged in panels C and E. Neurite projections (axons positively labeled with neurofilament antibodies, arrowheads in C and E) were observed among the implanted neurons. The EGFP fluorescence of some implanted DRG neurons was weak in D (arrows). However, these DRG neurons were positively labeled with neurofilament antibody (arrows in E and F). Scale bar shown in C and E: 30 Am. tion. A two-tailed, paired t test was used to determine whether the surviving DRG neurons were significantly influenced by the exogenous NGF. Results Identification and location of the DRG implants Mouse DRG implants were found in the rat cochleas for up to 6 weeks following the implantation (c.f., Table 1). The fluorescence of EGFP-positive DRG cells was readily identified in the implants (Figs. 2A, D). The DRG neurons were labeled by neurofilament antibody (Figs. 2B, E). In order to detect the subtype of the surviving DRG cells in the inner ear, the DRG neurons were further positively labeled with SP (Fig. 3A) and CGRP antibodies (Fig. 3B). Most DRG neurons were located in clusters, and single neurons were rarely seen. At 3 weeks following transplantation in the NGF group, the DRG implants were found in all rat cochleas (6/6, c.f., Table 1). All implants were attached to the osseous modiolus and close to the spiral ganglion region and the organ of Corti (Figs. 4 and 5). The DRG implants were also observed to penetrate through the basilar membrane towards the organ of Corti and the scala media (Fig. 4). At 6 weeks following implantation, DRG implants were found within the scala tympani and close to the spiral ganglion region (Fig. 6) in two host cochleas (2/6, c.f., Table 1) supplemented with exogenous NGF. A significantly lower survival was seen in the non-NGF groups. At 3 weeks following transplantation, DRG implants were found in four animals (4/6, c.f., Table 1). All implants were attached to the osseous modiolus and close to the spiral ganglion region (Fig. 7). At 6 weeks following transplantation, no DRG implants were found in the non-NGF group (0/6). Neurite outgrowth from the implanted DRG neurons and the neurite projections between the transplanted neurons and the host cochlear neurons At 3 weeks following transplantation in the NGF group, extensive neurite outgrowth from the transplanted DRG neurons was found following transplantation (Figs. 2 and 5). Furthermore, neurite projections (the axons positively labeled with neurofilament antibody, c.f., Figs. 2 and 5) were observed between the Fig. 3. Mouse DRG cells were labeled with substance P (SP, A), calcitonin gene-related peptide (CGRP, B) antibodies following transplantation into the adult rat inner ear with a supplement of NGF at 3-week survival. Scale bar: 30 Am. 188 Z. Hu et al. / Neurobiology of Disease 18 (2005) 184–192 Fig. 4. Implanted mouse DRG neurons survived close to the organ of Corti at 3 weeks following transplantation into rat cochlea with supplemented NGF. The DRG neurons were double-labeled with EGFP and neurofilament antibodies (arrow, shown in yellow), while host auditory nerve fibers were labeled with neurofilament and are shown in red. One DRG neuron was passing through the basilar membrane towards the organ of Corti (arrow, A, B). Two DRG neurons had already been located within scala media and close to the organ of Corti (arrow heads, A, B). The surviving DRG neurons also generated axons towards host cochlear nerve fibers and had reached to the bony modiolus (double arrows, A, C). Scale bar shown in A: 50 Am. implanted DRG neurons and the host SGNs (Fig. 5) as well as among the surviving DRG neurons (Fig. 2). At 3 weeks following transplantation, the neurite outgrowth from the DRG neurons was found to reach the osseous modiolus (Figs. 4A, C) and penetrate through the osseous modiolus towards the spiral ganglion region (Fig. 5). Furthermore, numerous neurofilament positive axons were observed between the implanted DRG neurons and the host SGNs in three animals supplemented with exogenous NGF (3/6, Fig. 5, c.f., Table 1). At 6 weeks following the implantation, the DRG neurons had axons extending towards the spiral ganglion region in the NGF group (Fig. 6). In the non-NGF group, the DRG neurons were found close to the modiolus in four animals (4/6), however, with relatively short neurite outgrowth from the DRG neurons as compared to the NGF group. No distinct neurite projections between the implanted DRG neurons and the host SGNs were observed at 3 weeks following transplantation among these animals (Fig. 7). Quantification of DRG neurons In the nontransplanted control DRGs (n = 6), the average neuronal number (mean F SD) in each DRG was 2178 F 225 with an average diameter of 17.7 F 4.0 Am. At 3 weeks after transplantation, the number of the implanted DRG neurons in the NGF group (332 F 36, 15% of control DRGs) was significantly higher than that in the non-NGF group (135 F 14, 6% of control DRGs; P b 0.01, two-tailed, paired t test, c.f., Table 1). The mean diameter of the surviving DRG neurons in the NGF group was 18.0 F 2.7 Am, while in the non-NGF group, this was 17.6 F 2.9 Am illustrating no significant difference. At 3-week survival, the average (mean F SD) length of neurite outgrowth of the surviving DRG neurons was 55.9 F 22.2 Am in the NGF group, while this was significantly shorter (14.8 F 7.4 Am) in the non-NGF group (P b 0.01, two-tailed, paired t test, c.f., Table 1). Discussion In this study, transplantation of mouse embryonic DRGs was combined with infusion of exogenous NGF into the adult rat cochlea. The results demonstrate that NGF promotes DRG neuronal survival and stimulates extensive neurite outgrowth from the surviving DRG neurons. The outgrowing DRG axons have been observed to penetrate the osseous modiolus towards the spiral ganglion region. Moreover, extensive neurite projections were found between the implanted DRG neurons and the host cochlear neurons. Our choice of donor tissue originates from our previous studies where we have transplanted DRG neurons into the adult cochlea and the cochlear nerve (Hu et al., 2004a,b; Olivius et al., 2003, 2004). It should be noted, however, that although DRGs have been successful in terms of neuronal survival and migration towards the SGNs and seem to have a potential for integration within the auditory neuronal system, DRGs may not be the only feasible alternative for replacing SGNs in the clinic. The best way to replace degenerated SGNs may be to implant with viable SGNs. However, it should be noted that our findings may also provide vital information regarding inner ear transplantation. This information may be relevant to implantation of various tissues, that is, SGNs and stem cells into the inner ear (Hu et al., 2004c). Neurotrophic factors are vital agents in neuronal development, including regulation of neuronal survival, affecting nerve fiber elongation and sprouting (Nosrat et al., 2001; Snider, 1994; Tuttle and O’Leary, 1998). In the auditory system, neurotrophic factors Z. Hu et al. / Neurobiology of Disease 18 (2005) 184–192 189 Fig. 5. Transplanted mouse DRG neurons were found survival close to the spiral ganglion region at 3 weeks following implantation into adult rat inner ear with the supplement of exogenous NGF. Extensive neurite outgrowth (axons positively labeled with neurofilament antibodies, arrow heads) was observed between the implanted DRG neurons (yellow, double-labeled with EGFP and neurofilament antibodies) and the host spiral ganglion neurons (SGN; red, labeled with neurofilament antibodies). b----Q indicates the bone separating the spiral ganglion region and the scala tympani. Scale bar: 30 Am. possess the capacity to prevent neuronal degeneration in deafened animals (Shah et al., 1995; Shinohara et al., 2002). In the present study, in order to enhance the survival of DRG neurons following transplantation, NGF was infused into the inner ear for a period of 2 weeks. At 3-week postoperative survival time, the implanted DRG neurons had a significantly higher survival in the NGF group than that in the non-NGF group (P b 0.01, c.f., Table. 1). At 6 weeks postoperatively, surviving DRG implants were found only in the NGF group (2/6), while no surviving DRG implants were found in the non-NGF group (0/6), precluding statistical analysis at this time point. This illustrates that exogenously supplemented NGF can enhance the DRG neuronal survival following transplantation into the adult inner ear. Furthermore, at 3 weeks following transplantation, it was observed that the DRG neurons in the NGF group had considerably longer neurite outgrowth than in the non-NGF group. Interestingly, the neurite outgrowth of the DRG neurons was observed to extend towards the SGNs. Thus, newly formed axons reached to the osseous spiral ganglion region (Fig. 4), having penetrated the bone and migrated close to the spiral ganglion region. In the animals supplemented with NGF, neurite projections were found between the implanted DRG neurons and the host SGNs (Fig. 5). In the non-NGF group, the DRG neurons exhibited significantly shorter neurite outgrowth than following NGF treatment (Fig. 7, c.f., Table 1, P b 0.01). In order to provide a functional neuronal replacement strategy into the inner ear, apart from survival, the migration and integration of the implant into the host auditory system are essential. In our model, we have seen migration of the implanted neurons not only through the modiolar bone towards the SGNs, but also in close association with the host cochlear neurons. We have previously demonstrated that the DRG neurons can migrate through the bone and become located within the spiral ganglion region (Hu et al., 2004b). Here, we further illustrate that, following supplement of exogenous NGF, the implanted DRG neurons send extensive neurite outgrowth towards the functionally important region in the cochlea, the spiral ganglion (Figs. 4 and 5). Interestingly, in this study, neurite projections were observed between the DRG neurons and the host SGNs. It is the first report to suggest the possibility of structural connections between the implanted neurons and the host cochlear neurons, which implies the potential of the transplanted neurons to replace the degenerated auditory neurons. It is therefore possible that the implanted DRG neurons can become integrated within the auditory system, thereby turning into a functional auditory unit. However, in order to achieve hearing function, a neural pathway from the SGNs towards the cochlear nucleus and further centrally towards the appropriate brain cortex areas must be present. We have recently found that the implanted DRG neurons survived and migrated centrally towards the brainstem following transplantation into the auditory nerve (Hu et al., 2004a). This indicates that implanted DRG neurons have the potential to extend axons towards a functionally appropriate location in the brainstem. SP and CGRP are well-known neuronal markers for the, respectively, small- and small-to-medium-sized DRG cells (Verge et al., 1990; Villar et al., 1989; Zhang et al., 1995). In the present study, we also found that the DRG neurons in the cochlea were stained positive for SP and CGRP antibodies. The axons of the small- and medium-sized DRG cells are composed of unmyelinated C and myelinated Ay axons. The conduction velocity of the Ay axon is 5–30 m/s, while the conduction velocity of the mammalian cochlear nerve is around 10–20 m/s (Moller et al., 190 Z. Hu et al. / Neurobiology of Disease 18 (2005) 184–192 Fig. 6. Mouse DRG neurons survived for 6 weeks following transplantation into adult rat inner ear in the NGF group. The inserted gray window illustrates the overview structure of the inner ear. The implanted DRG neurons (arrows, shown in yellow) were located within scala tympani and close to spiral ganglion neurons (labeled red). The DRG neurons were double-labeled with EGFP and neurofilament antibodies thus shown in yellow. The neurite outgrowth (arrow heads) from the implanted neurons was observed to extend towards spiral ganglion neurons. b----Q indicates the bone separating scala tympani and modiolus. Scale bar: 30 Am. 1994; Nguyen et al., 1999). However, it remains to be shown whether the DRG axons have a potential to replace the conduction function of the degenerated cochlear nerve. Neurite outgrowth from DRG neurons and the DRG neuronal migration were directed towards the spiral ganglion region in this study. We speculate that the implanted DRG neurons may utilize the tiny holes that have been shown to pass through the bony modiolus towards the spiral ganglion (Hu et al., 2004b; Lim, 1986; Lim and Kim, 1983). These small openings, Schuknecht’s canaliculae perforantes, would then provide a path for the DRG axons to Fig. 7. Mouse DRG implant survived 3 weeks following transplantation into the rat inner ear without exogenous NGF supplement. The inserted gray window illustrates the overview structure of the inner ear. The DRG neurons were located close to the spiral ganglion neurons and double-labeled with EGFP and neurofilament antibodies (shown in yellow, arrows). Relatively short neurite outgrowth was identified from these implanted neurons. The host auditory nervous system was labeled with neurofilament antibodies (shown in red). b----Q illustrates the bone separating the spiral ganglion region and the scala tympani. Scale bar: 50 Am. Z. Hu et al. / Neurobiology of Disease 18 (2005) 184–192 penetrate the bone and reach to the spiral ganglion region, possibly directed by neurotrophic factors released from the SGNs (Hansen et al., 2001; Rubel and Fritzsch, 2002). The DRG neurons were also observed to penetrate the basilar membrane and migrate towards the organ of Corti (c.f., Figs. 4A, B). It is therefore likely that the DRG neurons were directed by endogenous growth factors released by the hair cells (Qun et al., 1999; Rubel and Fritzsch, 2002). In summary, the present findings demonstrate that xenografted embryonic DRG neuronal implants survive for up to 6 weeks after transplantation into the adult cochlea with the supplement of NGF. Extensive neurite outgrowth from the DRG neurons was identified towards the functionally important area in the inner ear, that is, the spiral ganglion region. Neurite projections between the transplanted DRG neurons and the host cochlear neurons were also observed in this study, suggesting the possibility that DRG implants may become part of the functionally auditory system. In future studies, the feasibility of the implanted DRGs forming synapses with the host auditory system, releasing appropriate neurotransmitters, and becoming functionally integrated within the auditory system should be investigated. In combination with a cochlear prosthesis, this neuronal implantation strategy may provide possibilities for future treatment of profoundly deaf patients. Acknowledgments The authors would like to thank Dr. Leif J7rlebark and Dr. Anders Fridberger for helpful comments on the manuscript. This study was supported by Petrus and Augusta Hedlund Foundation, The Swedish Association of Hard of Hearing People, The Ollie and Elof Ericssons Foundation for Medical Research, Acta Otolaryngologica’s foundation, The Foundation Tysta Skolan, 2ke Wiberg’s foundation, Magnus Bergvall’s foundation, The Swedish Insurance Society, The Foundation Goljes Minne, The Swedish Research Council (09888), the Karolinska Institutet, and the European Commission (contract no. 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