BR A IN RE S E A RCH 1 3 77 ( 20 1 1 ) 4 1 –4 9 available at www.sciencedirect.com www.elsevier.com/locate/brainres Research Report Horseradish peroxidase dye tracing and embryonic statoacoustic ganglion cell transplantation in the rat auditory nerve trunk Björn Palmgrena,b,⁎, Zhe Jinc , Yu Jiaoa,d , Beata Kostyszyna , Petri Oliviusa,b,e a Center for Hearing and Communication Research, Karolinska University Hospital, 171 76, Stockholm, Sweden Department of Clinical Sciences, Intervention and Technology (CLINTEC), Section of Otorhinolaryngology, Karolinska Institutet, Karolinska University Hospital, 171 76 Stockholm, Sweden c Department of Neuroscience, Uppsala University, Box 593, 751 24, Uppsala, Sweden d Department of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, 100730, Beijing, China e ENT clinic, Linköping University hospital, 58185 Linköping, Sweden b A R T I C LE I N FO AB S T R A C T Article history: At present severe damage to hair cells and sensory neurons in the inner ear results in non- Accepted 28 December 2010 treatable auditory disorders. Cell implantation is a potential treatment for various Available online 6 January 2011 neurological disorders and has already been used in clinical practice. In the inner ear, delivery of therapeutic substances including neurotrophic factors and stem cells provide Keywords: strategies that in the future may ameliorate or restore hearing impairment. In order to Auditory nerve describe a surgical auditory nerve trunk approach, in the present paper we injected the Horseradish peroxidase neuronal tracer horseradish peroxidase (HRP) into the central part of the nerve by an intra Statoacoustic ganglion cranial approach. We further evaluated the applicability of the present approach by Cell transplantation implanting statoacoustic ganglion (SAG) cells into the same location of the auditory nerve in Transitional zone normal hearing rats or animals deafened by application of β-bungarotoxin to the round window niche. The HRP results illustrate labeling in the cochlear nucleus in the brain stem as well as peripherally in the spiral ganglion neurons in the cochlea. The transplanted SAGs were observed within the auditory nerve trunk but no more peripheral than the CNS-PNS transitional zone. Interestingly, the auditory nerve injection did not impair auditory function, as evidenced by the auditory brainstem response. The present findings illustrate that an auditory nerve trunk approach may well access the entire auditory nerve and does not compromise auditory function. We suggest that such an approach might compose a suitable route for cell transplantation into this sensory cranial nerve. © 2011 Elsevier B.V. All rights reserved. ⁎ Corresponding author. Karolinska University Hospital, 171 76 Stockholm, Sweden. Fax: +46 851774265. E-mail address: bjorn.palmgren@karolinska.se (B. Palmgren). Abbreviations: ABR, auditory brainstem response; β-BuTx, β-bungarotoxin; BS, brainstem; CN, cochlear nucleus; CNS, central nervous system; CSF, cerebrospinal fluid; E13, embryonic day 13; EDTA, ethylenediaminetetraacetic acid; FBS, foetal bovine serum; GFP, green fluorescent protein; HRP, horseradish peroxidase; IAM, internal auditory meatus; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PNS, peripheral nervous system; RW, round window; SAG, statoscoustic ganglion; SGN, spiral ganglion neuron; TMB, tetramethylbenzidine; TZ, transitional zone 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.12.078 42 1. BR A IN RE S EA RCH 1 3 77 ( 20 1 1 ) 4 1 –49 Introduction Within the peripheral hearing organ, i.e. the cochlea, the specialized hair cells and neurons are major targets of both intrinsic genetic changes (e.g. gene mutation) and exogenous insults (e.g. noise and pharmacological trauma) that consequently result in hearing impairment. In order to restore or even replace the degenerated cells in the cochlea different substrates such as pharmacologic agents, viral vectors, mature or immature cells have been delivered into the cochlea using a range of surgical techniques (Kesser and Lalwani, 2009; Regala et al., 2005; Richardson et al., 2006; Sekiya et al., 2007b). Several commonly performed surgical approaches to access the cochlea (e.g. cochleostomy) may disturb the intracochlear structure and jeopardize residual hearing. Application of therapeutic substances (e.g. steroids) to the round window membrane presents a non-traumatic approach in the prevention or treatment of certain reversible inner ear diseases (Arriaga and Goldman, 1998; Silverstein et al., 1999). The permeability of the human round window membrane to each therapeutic agent is not yet fully explored (Carvalho and Lalwani, 1999). In addition, the majorities of surgical routes to access the cochlea are performed in smaller experimental animals but are not yet available in humans. There are also indications that axons from cells transplanted into the peripheral portion of the cochlear nerve may be inhibited by the transitional zone (TZ) located between the central nervous system (CNS) and the peripheral nervous system (PNS), thereby precluding any further central sprouting (Fraher, 2000; Sekiya et al., 2007b). In order to improve the cell delivery process novel approaches with a potential to counteract irreversible damage to spiral ganglion neurons (SGNs) including degeneration of the auditory nerve, might be essential. Only a few experimental studies have adopted the approach to deliver cells or substances to the central portion of the auditory nerve (Corrales et al., 2006; Sekiya et al., 2006, 2007a). In the current paper we describe a suboccipital approach to initially inject the neuronal tracer horseradish peroxidase (HRP) into the rat auditory nerve. The function of the auditory nerve pre- and two weeks postoperatively was monitored by measuring the auditory brainstem response (ABR). In order to assess the applicability of this surgical approach, mouse statoacoustic ganglion (SAG) explants were implanted using the same approach. Our findings illustrate that the HRP-tracer injected into the rat auditory nerve trunk by the internal auditory meatus (IAM) was transported to the central as well as peripheral portions of the nerve. Furthermore the ABR measurements demonstrated that the surgical procedure did not compromise auditory function. We also illustrate that the transplanted SAG explants can survive in the auditory nerve for up to five weeks, though in reduced numbers. 2. Results 2.1. HRP-tracer distribution after auditory nerve injection The neuronal tracer HRP was used to verify the precision and the distribution of the suboccipital approach injection into the auditory trunk (Table 2). Depending on the volume of the injected substance there will also be a certain amount of extra cellular HRP proximal to the injection site as observed by accumulation of the blue TMB reaction product (Fig. 2). Here, the accumulation of HRP-tracer around the injection site was easily identified (Fig. 2A, B). Further, the tracer was transported centrally as well as peripherally from the injection site (Figs. 2A, B). In the peripheral portion the HRP labeling was observed in the auditory nerve trunk (Fig. 2a1), in the Schwann-glial junctional zone of the auditory nerve (Fig. 2a2) and in the spiral ganglion neurons (Fig. 2b1 and b2). In the central portion HRP was found in the central SGN terminals in the cochlear nucleus (Fig. 2b3). No HRP labeling was observed in the contralateral ear thereby making the possibility of leakage of HRP tracer from the injection side unlikely. 2.2. nerve Transplantation of SAG-GFP cells into the auditory In both the non-deafened and the β-bungarotoxin-deafened rats (survival times two to five weeks, groups 2–5, Table 1) we identified transplanted SAGs at the injection site as well as Fig. 1 – (A). Experimental setup for intra-auditory nerve trunk delivery. A Hamilton syringe attached with a 33-gauge needle was mounted in the clamping device of the stereotactic frame. (B). The injection site at the auditory nerve root (arrow) and the injection needle (arrowhead). BR A IN RE S E A RCH 1 3 77 ( 20 1 1 ) 4 1 –4 9 43 Fig. 2 – HRP distribution (visualized as blue TMB reaction product) after injection to the rat auditory nerve trunk. The sections A and B are from different animals. HRP labeling can be found in the auditory nerve trunk (A and a1), in the Schwann-glial junctional zone (arrowhead) of the auditory nerve (A and a2), in the spiral ganglion neurons (B and b1 and b2). In the central portion HRP was found in the cochlear nucleus (B and b3). Arrow, blue TMB reaction product; AN, auditory nerve; CN, cochlear nucleus; SGN, spiral ganglion neuron; PM, peripheral myelin; CM, central myelin. Star, injection site. Scale bar: A, B: 100 μm; a1–a2, b1–b3: 40 μm. along the auditory nerve (Fig. 3). In two animals we detected both GFP- and Tuj1-positive cells with detectable neurites in the nerve peripherally from the IAM (Figs. 3E and F). We also observed GFP-positive cell profiles lining the boundary of the Schwann-glial TZ (Fig. 4C) but we did not observe any cells passing through the TZ towards the periphery of the auditory nerve. Furthermore there were no transplanted cells migrating centrally into the cochlear nucleus. In order to illustrate that our observed GFP-positive profiles (stained with anti-GFP antibody) are transplanted cells but not artifacts e.g. autofluorescence from blood or immune cells migrating from the injection site, we performed sham surgery by injecting culture medium only by the same approach. In these control animals we did not observe any cells stained with anti-GFP antibody (Fig. 4B). In the deafened groups (4 and 5) we found cell profiles in five out of eight animals whereas among the non deafened animals (groups 2 and 3) cell profiles were found in one out of nine animals only. In some animals we observed GFP positive tissue without cell profiles (data not shown). In four animals we could not detect any GFP-positive profiles at all. Two animals from the deafened groups had to be sacrificed due to wound infections. 44 BR A IN RE S EA RCH 1 3 77 ( 20 1 1 ) 4 1 –49 Table 1 – Groups of rats used either for SAG implantation or HRP injection to the auditory nerve trunk. Group Number of animals (n) HRP injection to AN SAG transplantation to AN RW application of β-BuTx Survival time 1 2 3 4 5 6 2.3. 6 6 3 4 6 3 + − − − − − − + + + + − (only culture medium) Assessment of auditory function Immediately before and two weeks after intra auditory nerve HRP injection ABR was measured at 8, 16 and 40 kHz. The magnitudes of the ABR threshold shifts pre- as compared to postoperatively were approximately 5 dB at all frequencies measured, illustrating no statistically significant differences between the pre- and post operative ABR values (Fig. 5). In two out of six animals the ABR thresholds were not altered postoperatively at all. 3. Discussion Studies of transplanted stem cells into the cochlea or the cochlear nerve have not been able to visualize significant numbers of newly-formed neuronal connections between the implant and the cochlear nucleus in the brain stem. In the present paper, in order to explore differentiation but also migration of the implanted progenitor cells the aim was to illustrate a technique for injection of cells into the central portion of the auditory nerve. Furthermore, by injecting HRP tracer into the auditory nerve trunk we suggest that the injection technique allows injected trophic factors or other substances to reach into the cochlear nucleus region in the brain stem and also into the SGN in the cochlea. By measuring the ABR-response we assessed whether the implantation procedure would have any impact on auditory function. Finally, by using the same suboccipital approach as for the HRP injection, we implanted embryonic mouse SAG explants into the auditory nerve. Being a widely used tracer for neuronal pathways (van der Want et al., 1997; Waar et al., 1981) we selected HRP for the tracing procedure used in this paper. Since the HRP uptake occurs mainly by passive endocytosis in the axotomized regions and nerve terminals (van der Want et al., 1997) we presume that the trauma on the auditory nerve trunk caused by the injection needle would be permissive for uptake of the substrate. Seemingly, in accordance with our results other Table 2 – The distribution of HRP following auditory nerve trunk injection by the internal auditory meatus. HRP positive staining area Number of cochleas Auditory nerve trunk Schwann-glial junctional zone Spiral ganglion neurons Cochlear nucleus 6 4 5 5 − − − + + + 2 days 2 weeks 5 weeks 2 weeks 5 weeks 2 weeks studies have shown that following pressure injection there are two different types of HRP uptake into the neurons. Apart from the local accumulation of HRP there is a diffuse passive uptake that could be due to HRP pressure in the confined injection site. The second type of uptake is the physiological incorporation with transportation and diffusion of HRP by the intact axonal terminals (Leake-Jones and Snyder, 1982). We found HRP labelings were located by the injection site, in SGN in the cochlea and in central terminals in the cochlear nucleus (CN) close to the second order neurons. This verifies the injection site and illustrates that the injection procedure would reach into target areas we presume would be relevant for a successful outcome of an implantation paradigm. We further speculate that such a paradigm might have a potential to promote survival of implanted cells to differentiate and send afferents into the CN in the BS as well as connecting with hair cells in the cochlea. Locally applied growth factors could also be distributed to the auditory nerve by diffuse uptake as well as transported peripherally and centrally. We did not observe any HRP labeling in the contralateral ear indicating that the HRP-tracer did neither leak into the CSF nor spread contralaterally via the systemic blood circulation. Stem cells are present in the rat embryonic inner ear but decrease in numbers post partum (Oshima et al., 2007). This is probably one reason for the poor ability of the inner ear to regenerate damaged spiral ganglion neurons and hair cells. The SAG explants used in the present experiment were harvested from the auditory tract in E13 embryos. At this time period neuronal responses to sound initializes (Friauf, 1992; Uziel et al., 1981). The SAGs contain embryonic progenitor cells responsible for the development of both cochlear and vestibular neurons (Sher, 1971). Earlier studies have shown that histological signs of severe rejection appears following transplantation of cells to non-immunosuppressed rats (Fernandez et al., 2006). All animals in our experiments received daily injections of immunosuppressants and antibiotics after the cell transplantation during the entire survival time. In comparison to the distribution of the HRP tracer the transplanted SAG cells were not observed at longer distances away from the injection site. Furthermore, although we observed survival of implanted explants for up to five weeks these were only found in relatively small numbers. This could be due to several reasons but we speculate that, in order to survive in larger numbers, the injected cells might need exogenous neurotrophic support. Examples of neurotrophic factors to improve implant survival would be brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF) and neurothrophin-3 (NT-3) that have been shown to increase SGN survival (Ernfors et al., 1995). In the beginning of BR A IN RE S E A RCH 1 3 77 ( 20 1 1 ) 4 1 –4 9 45 Fig. 3 – Survival of SAG cells after transplantation to the rat auditory nerve trunk. Photomicrographs A and B were taken from non-deafened rats with 5 weeks postoperative survival time, C-F from β-bungarotoxin-deafened rats with 2 weeks postoperative survival time. Single GFP-positive SAG cells (green color) (A, B and inset in B; arrow) and GFP-positive cell clusters (C, D; asterisk) were found in the auditory nerve. GFP- and TUJ1- (red color) double stained SAG cells (yellow color) with distinct neurites (E, F; arrowhead) can also be detected in the auditory nerve. The nuclei were counterstained with DAPI (blue color). AN, auditory nerve; CN, cochlear nucleus; M, cochlear modiolus. Star, injection site. Scale bar: A, 200 μm; B, 100 μm; C and E 400 μm; D, F and inset in B, 50 μm. the synaptogenesis it has been shown that the cochlear neurons are mainly dependant on NT-3 whereas the vestibular neurons are more dependent on BDNF (Fritzsch et al., 1997). Other studies have shown that, for proper survival, migration and differentiation, the early SAG neurons are also dependent on BDNF and fibroblast growth factors (FGFs) (Brumwell et al., 2000; Hossain et al., 1997). Some technical problems, possibly precluding implant survival, were encountered involving the easily disrupted well-vascularized areas by the IAM close to the auditory nerve. Furthermore, we only examined for any potential neurite outgrowth during a five week postoperative period whereas it cannot be excluded that the development of newly-formed neuritic projections would require a longer survival time. We did not observe any SAG cells in the cochlear perilymph or endolymph indicating that there was no cell leakage via the CSF or via the canaliculi perforantes in the cochlear modiolus. In previous studies we have interestingly observed migration 46 BR A IN RE S EA RCH 1 3 77 ( 20 1 1 ) 4 1 –49 Fig. 5 – ABR threshold in non-deafened rats before and 2 weeks after HRP injection. Fig. 4 – Migration of transplanted SAG cell profiles to the Schwann-glial junctional zone. The location of Schwann-glial junctional zone (A). GFP-positive cells (arrow) were detected along the boundary of the Schwann-glial junctional zone (arrowhead) in the animals with SAG explants transplanted to the auditory nerve (C and inset in C), but not in the sham-operated animals with culture medium injection (B). (B) and (C) were shown from the rectangular area in (A). The nuclei were counterstained with DAPI (blue color). AN, auditory nerve; PM, peripheral myelin; CM, central myelin. Scale bar: A, B and C, 50 μm; inset in C, 10 μm. of embryonic sensory cells from the perilymph to the SGN in the modiolus following implantation into the inner ear scala tympani (Hu et al., 2004a; Olivius et al., 2003). We suggested that these migrating cells might utilize the canaliculi perforantes (Hu et al., 2004b). Furthermore, in the present paper we did not observe any cells in the contralateral cochlear specimen even though this does not completely rule out a possible route for cell migration via the CSF into the cochlear aqueduct. We speculate that since our SAG cells were not dispersed in the injected medium but ensheathed with fibrous tissue in whole explants this may reduce the ability of the SAGs to migrate and send out neurites. In some specimen we found GFP-positive tissue without any cells. This could either be because the transplanted SAG explants did not contain sufficient numbers of cells or that these did not survive in sufficient numbers. In terms of SAG cell survival there was a significant difference between the non-deafened and β-bungarotoxin-deafened groups. The lower survival-rate of cells in the non-deafened group could be due to similar mechanisms indicated by previous studies, e.g. that the migration of implanted cells in nerves is limited by the available space in the nerve (Sekiya et al., 2006). Other explanations for the limited cell migration might be the CNS-PNS TZ. This border zone between the CNS and PNS, illustrating a Schwann cell–glial cell barrier, represents a biological obstacle for various molecules and cells reaching into the CNS (Fraher, 2000). Subsequently we hypothesize that the TZ might also hamper the migration of larger molecules and cells. We are currently investigating the possibility to make, by injecting selected substances or supporting cells together with the SAGs, the TZ more permeable and potentially facilitate migration and sprouting of implanted cells. One possible problem by using an implantation approach directed towards the auditory nerve is that it might compromise the integrity of the cochlea and the hearing. In the present study, however, as evaluated from our fairly unaltered ABR curves the nerve trunk approach does not seem to significantly impair auditory function. In summary, the present study illustrates that the surgical approach presented can be useful in reaching the SGN soma including their central terminals, e.g. the entire auditory nerve. The findings also suggest that the approach may hold the promise to reach regions in the auditory nerve seemingly relevant for a successful implantation outcome without compromising hearing. We further suggest that the similar stereotactic setup may be used for delivery of neurotrophic factors essential to implant survival and differentiation. Such studies are under way. 4. Experimental procedures All animal experiments followed the national approved protocol for care and use of animals in Sweden (approval N58/03, N347/05). Young adult Sprague–Dawley rats (n = 28; 200–250 g) were used in the study. The different animal groups are presented in Table 1. Preoperative otoscopic BR A IN RE S E A RCH 1 3 77 ( 20 1 1 ) 4 1 –4 9 examinations were performed to exclude any visible middle ear infection. 4.1. Surgical approach and HRP injection HRP animals (n = 6) were anaesthetized with an intraperitoneal (i.p.) injection of a mixture of Ketalar© (50 mg/kg) and Rompun© (10 mg/kg) and placed in a stereotactic frame. The skull was put in a fixed position and the skin on the occipital region shaved and disinfected with 70% ethanol. Under a surgical microscope a left post-occipital hemi-arcade incision was made through the skin and underlying soft tissue. By using a drill a 3 mm diameter hole was made on the suboccipital bone. By sharp incision the underlying dura was opened and reflected towards the edge of the hole followed by drainage of cerebrospinal fluid (CSF). As part of the posterolateral cerebellar hemisphere was gently retracted contralaterally a cotton ball was placed for CSF suction thereby revealing the auditory nerve trunk between the brainstem and the internal auditory meatus. A 10 μl Hamilton syringe attached with a 33-gauge needle was filled with 30% HRP (Type VI-A, Sigma) and mounted in the clamping device of the stereotactic frame (Fig. 1A). The needle was positioned above the auditory nerve trunk with the angle of the tip adjusted towards the internal auditory meatus. The needle was lowered into the auditory nerve trunk with a depth of 500 μm by the use of the micromanipulator (Fig. 1B). A total volume of 4 μl of HRP solution was injected into the nerve root. After injection the needle was left in place for 10 min whereafter the wound cavity was filled with sterile saline. A piece of fascia was used for covering the hole in the dura and occipital bone. The wound was closed in layers with continuous single sutures. Following removal from the stereotactic frame the animals were given subcutaneous injections of 3 ml saline and 0.2 ml Temgesic© (0.3 mg/ml) and placed in a warm cage to recover before being transferred to the home cage. 4.2. HRP histochemical staining Two days following injection the HRP animals were deeply anaesthetized with an intraperitoneal overdose of pentobarbital (60 mg/ml) and transcardiacally perfused with 0.9% NaCl followed by 4% of paraformaldehyde (PFA). Following decapitation, the left temporal bone, auditory nerve and adjacent brainstem were carefully excised in a single tissue block. The temporal bone was opened and the excess bony tissue removed. Under a dissecting microscope the cochlea was perfused with 4% PFA in 0.1 M PBS through the round window and a hole was made in the apical turn. The tissue block was immersed in fixative for 24 h at 4 °C and washed by PBS. Decalcification with 0.1 M ethylenediaminetetraacetic acid (EDTA) in 0.1 M PBS at 4 °C was carried out on the whole tissue block until the remaining bony tissue was soft enough for cryostat sectioning. The tissue block was immersed in 20% sucrose for 24 h, embedded in optimal cutting temperature (OCT) compound (Sakura Tissue-Tek) and 12 μm serial cryostat sections were made. The cryostat sections were processed for HRP using tetramethylbenzidine (TMB) as the chromagen and sodium tungstate (ST) as the stabilizer (Gu et al., 1992). In brief, sections were dried at room temperature (RT) for 2 h, rinsed three times 47 during 10 min in PBS and pre-incubated in reaction medium (0.5% TMB in ethanol and 1% ST in 0.2 M PBS) at RT for 20 min while protected from light. The reaction was initiated by adding 0.7 ml of 0.3% hydrogen peroxide every 10 min during the 1 h incubation period. To terminate the reaction the sections were rinsed 5 times for 3 min in 0.05 M PBS (pH 5.0–5.4). All sections with or without eosin counterstaining were dehydrated through ethanol series, cleared in xylene, mounted with Permount and photographed using a light microscope (Zeiss) equipped with a digital camera (Nikon Coolpix 990). Negative controls were made by omission of TMB in the sections. 4.3. niche Application of β-bungarotoxin to the round window Animals (n = 13) were deafened by application of β-bungarotoxin (β-BuTx) to the round window niche as described previously (Palmgren et al., 2010). In brief, after i.p. anaesthesia with xylazine (10 mg/kg i.p.) and ketamine (50 mg/kg i.p.) the round window niche was exposed by a retroauricular incision. 5 μl of β-BuTx (0.05 μg/ml, Alexis Biochemicals) was absorbed by gel foam and applied to completely fill the round window niche. A piece of fascia was placed to cover the hole in the bulla. The animals were kept for 3 weeks until further surgical procedures were performed. 4.4. Transplantation of statoacoustic ganglion explants to the rat auditory nerve SAG explants dissection was performed in embryonic day 13 (E13) green fluorescent protein (GFP)-positive BalbC mice in Hanks Balanced Salt Solution (HBSS) supplemented with antibiotics. Whole explants were placed into the 4-well cell culture plates coated with poly-l-lysine and laminin. The SAG explants were cultured overnight in culture media consisting of Dulbecco's Modified Eagle's Medium (DMEM)/F12 (Gibco/ Invitrogen) supplemented with 1% Foetal Bovine Serum (FBS), Insulin-transferrin-sodium selenite supplement (ITSS), 4-(2Hydroxyethyl) piperazine-1ethansulfonic acid (HEPES) and antibiotics. The explants were removed from the cell culture plates with a needle and immediately used for implantation. The host rats had previously been anaesthetized with an intraperitoneal injection of a mixture of Ketalar© (50 mg/kg) and Rompun© (10 mg/kg) and the surgery was carried out by the suboccipital approach described above. The SAG explants were aspirated from a petri dish with a 10 μl Hamilton syringe. By using the same needle, the stereotactic frame and a syringe clamping device (Fig. 1B) the SAG explants were injected together with 4 μl of medium into the auditory nerve by the IAM. The needle was kept in place for 10 min after injection whereafter the wound was closed as above. Sham operated animals were injected with 5 μl culture medium. To prevent postoperative infection and immune response rejection all animals received daily doses of tetracycline (1.8 mg/ml, i.p.) and cyclosporine (4.2 mg/ml, i.p.). After the survival period the rats were sacrificed by an overdose of pentobarbital (60 mg/ml, i.p.), transcardially perfused with body warm 0.9% saline followed by ice-cold 4% PFA in 0.1 M PBS. The cochlea, auditory nerve and part of the brainstem were carefully removed en bloc. 48 4.5. BR A IN RE S EA RCH 1 3 77 ( 20 1 1 ) 4 1 –49 Immunohistochemistry The specimens (cochlea plus auditory nerve including brain stem) were dissected out and a small hole used for perfusion with PFA (initially 4% and then 0.5%) was made in the apex. The cochlea was decalcified in EDTA for 7 days. After 24 h in 20% sucrose solution the specimens were embedded and frozen in OCT Compound (Tissue-Tek; Sakura Finetek, Torrance, CA, USA). The specimens were orientated in the compound so that mid-modiolar sections would contain the cochlea, auditory nerve and brain stem (BS) as a continuum. The 12 μm mid-modiolar cryosections were mounted on glass slides. The sections were blocked with 10% goat serum, 5% bovine serum albumin (BSA) and 0.2% Triton X-100 in 0.1 M PBS for 1 h at room temperature and incubated for 36 h at 4 °C with conjugated goat polyclonal to GFP (FITC conjugated) antibody (1:200; Abcam, Cambridge, UK). Following incubation the samples were washed in PBS and put into blocking solution for 1 h at room temperature. For double staining the sections were incubated for 48 h at 4 °C with rabbit polyclonal β-tubulin (TUJ1) antibody (1:200; Covance Research Products, Berkeley, CA, USA). Following incubation the sections were labeled with goat-anti rabbit-Cy3 (1:2000) for 1 h at room temperature. The specimens were visualized and photographed using a fluorescence microscope (Zeiss, Stockholm, Sweden) equipped with a digital camera (Nikon Coolpix 990, Solna, Sweden). Omission of the primary antibody served as negative control. Cell nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI). 4.6. Auditory function assessment The ABR measurements (n = 6) were conducted under general anaesthesia with ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) immediately before and two weeks after surgery on the left ear in a soundproof booth using a Tucker-Davis System II (BioSig stimulate/recording system 2.0, Tucker-Davis Technologies, Alachua, FL, USA). The stimulus intensity was calibrated with a 0.25-inch condenser microphone (model 4135, Brüel & Kjær, Nærum, Denmark). All sound pressure levels were expressed in decibel values relative to 20 μPa. Sound stimulation (tone burst 20 stimuli/ s; single sinusoidal wave) was applied to the left ear using a high frequency transducer via a flexible tube in the external auditory meatus. Needle electrodes were placed on the vertex and below the recorded ear whereas the ground electrode was placed on the hind leg. 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