Introduction

Sudden hearing loss, which often results from disruption of cochlear function, is devastating to patients in the prime of life and is thought to be caused by an acute interruption of the blood supply to the inner ear.^1,2,3 However, the details of the effects of transient ischemia on the cochlea remain unclear. Using a technique called experimental hindbrain ischemia,⁴ we successfully made a chronic animal model of transient cochlear ischemia in Mongolian gerbils and demonstrated that inner ear damage was closely related to the progressive inner hair cell (IHC) loss that follows ischemia.^5,6,7 Therefore, rescuing the ischemia-induced degeneration of IHCs should be a way to save useful hearing after sudden deafness.

Adenovirus has recently attracted a great deal of attention as a vector for gene therapy. As a result of the anatomical and immunological isolation of the inner ear, cochlear cells appear to be good targets for gene transfer.^8,9,10 Studies of the role of genes in sensory hearing in vivo suggest that glial-cell-derived neurotrophic factor (GDNF) is one of the most effective therapeutic agents for preventing human auditory degeneration induced by acoustic overstimulation or ototoxic agents.^11,12 This study examined whether inoculation with Ad-GDNF vector caused significant overexpression of GDNF protein in the cochlea and prevented the progressive IHC damage induced by transient ischemia in our animal model.

Results

Distribution of the reporter transgene

The activity of the adenovirus vector was assayed histochemically. Six Ad-LacZ-inoculated animals were killed 4 days after inoculation. Three of these cochleae were used for whole-mount staining (Figure 1) and the other three were used for cryostat-section staining (Figure 2). LacZ-positive cells were seen throughout the cochlea, from the basal to the apical turn (Figure 1a), and the most conspicuous blue regions in the stereoscopic photograph corresponded to the lateral wall tissue (Figure 1b). A light photomicrograph of a 10-m-thick cryosection showed LacZ-positive cells in the mesothelial cell layer of the organ of Corti (arrow), Reissner membrane (double arrow) (Figure 2a), spinal ganglion cells (arrowhead), and around hair cells (double-headed arrow) (Figure 2b).

Figure 1.

Stereoscopic photomicrograph of X-gal staining. A stereoscopic photomicrograph of a cochlea obtained 4 days after administering Ad- Lac Z (a). After X-gal staining, transfected cells with blue cytosol are seen throughout the cochlea from the basal to the apical turn. The most conspicuous blue regions correspond to the mesothelial cells of the scala tympani and lateral wall tissue. A whole-mount specimen from a cochlea processed 4 days after administering Ad-Lac Z (b). All the staining-positive cells with blue cytosol are located in the supporting cell layer of the organ of Corti beneath the basilar membrane.

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Figure 2.

Cryosection of X-gal staining. A light photomicrograph of a 10-m-thick cryosection obtained 4 days after administering Ad- Lac Z shows positive-stained cells located in the mesothelial cell layer of the organ of Corti (arrow), Reissner membrane (double arrow) (a), spinal ganglion cells (arrowhead), and around hair cells (double-headed arrow) (b).

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GDNF protein production

In this study, viral inoculation was done 4 days before ischemia and the effects of Ad-GDNF vector were detected 7 days after ischemia. First, to evaluate the GDNF protein production when we induced ischemia, 18 animals were examined 4 days after the injection of Ad-GDNF, Ad-LacZ, or artificial perilymph (n=6 for each group). Figure 3 shows a representative Western blot showing a single GDNF band between the 30- and 46-kDa bands using GDNF-specific primary antibody¹³ in all groups. No band was detected when the blots were incubated without primary antibody. The results of the densitometric analysis are plotted in Figure 4. No significant change in the GDNF protein level was seen in the Ad-LacZ or artificial perilymph groups. In contrast, the GDNF protein level increased by more than four times in the Ad-GDNF group 4 days following inoculation.

Figure 3.

Western blot band of GDNF protein production. Representative Western blot showing a GDNF band to evaluate the GDNF protein production when we induced ischemia insult 4 days after injection of Ad-GDNF, Ad-LacZ, or artificial perilymph. A single band of GDNF protein is seen between the 30- and 46-kDa bands in all groups. No band was detected when the blots were incubated without primary antibody.

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Figure 4.

Densitometric analysis of GDNF protein production. The integrated optical density was obtained from digitized Western blot data using the program NIH Image. The data were normalized to internal standards (no-treatment animals) on each gel and given as percentage values. No significant change in the GDNF protein level was seen in the Ad-LacZ or artificial perilymph groups. In contrast, there was a significant increase in GDNF immunoreactivity in the Ad-GDNF group 4 days following inoculation (^*P<0.01).

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Next, to confirm the duration of GDNF protein production by the Ad-GDNF vector, 24 animals were examined 0, 4, 8, and 11 days after inoculation (n=6 for each time). Figure 5 shows a representative Western blot for Ad-GDNF vector. The densitometric analysis shows a significant increase in GDNF protein following inoculation with Ad-GDNF vector throughout the experiment (Figure 6).

Figure 5.

Western blot band of GDNF protein production by Ad-GDNF. Representative Western blot showing a GDNF band to confirm the duration of GDNF protein production by Ad-GDNF vector; 24 animals were examined 0, 4, 8, and 11 days after inoculation. A single band of GDNF protein is seen between the 30- and 46-kDa bands. No band was detected when the blots were incubated without primary antibody.

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Figure 6.

Densitometric analysis of the duration of GDNF protein production by Ad-GDNF. The integrated optical density was obtained from digitized Western blot data using the program NIH Image. The data were normalized to internal standards (no-treatment animals) on each gel and given as percentage values. The result shows a significant increase in the GDNF protein level following the inoculation of the Ad-GDNF vector until 11 days following inoculation.

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Toxicity of the virus vector

To evaluate the toxicity of the virus vector, compound compound action potential (CAP) thresholds and hair cell damage were measured after injection of Ad-GDNF vector, Ad-LacZ vector or artificial perilymph into the cochlea at the rate of 0.5 l/min for 4 min. The sequential changes in the CAP thresholds are summarized in Figure 7. There were no significant differences among the three groups, suggesting that the virus vector had no toxic effects electrophysiologically. Furthermore, the rates of inner hair cell (IHC) and outer hair cell (OHC) damage 11 days after injection are summarized in Figure 8. No cell damage was detected in any of the groups. These results show that at the dose used in this experiment, the virus vector had no toxic effects physiologically or histologically up to 11 days after virus inoculation.

Figure 7.

Changes in the CAP threshold after virus inoculation. CAP threshold after injection of Ad-GDNF vector, Ad-LacZ vector, or artificial perilymph at a rate of 0.5 l/min for 4 min into the cochlea. The CAP threshold before vertebral artery occlusion was defined as 0 dB. There were no significant differences among the three groups, suggesting that the virus has no toxic effects. All values are presented as the means.d. Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.

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Figure 8.

The rates of inner and outer hair cell loss 11 days after injection of Ad-GDNF vector, Ad-LacZ vector, or artificial perilymph. No cell damage was detected in any group. These results show that at the dose used in this experiment, the virus vector had no toxic effects, even 11 days after virus inoculation.

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Effect of the virus vector

The effect of the virus vector was determined by measuring the shift in the CAP thresholds (Figure 9). Viral inoculation was performed 4 days before ischemia. In all groups, occlusion of the vertebral arteries bilaterally caused a tremendous increase in the CAP threshold, which exceeded 120 dB SPL, the maximal output intensity of our measuring system. In both the Ad-LacZ and artificial perilymph groups, the CAP threshold did not return to pre-ischemic levels after ischemia. The average increase in the CAP threshold on the seventh day after ischemia was 17.1 and 18.3 dB in the Ad-LacZ and artificial perilymph groups, respectively. In the Ad-GDNF group, the CAP threshold recovered to the pre-ischemic level within 1 day after reperfusion and remained constant until the seventh day after ischemia. The average increase in the CAP threshold on the seventh day was significantly lower in the Ad-GDNF group than in the Ad-LacZ and artificial perilymph groups (P<0.01).

Figure 9.

Changes in the CAP threshold following transient cochlear ischemia. The CAP threshold before vertebral artery occlusion was defined as 0-dB. In the Ad-GDNF group, the CAP threshold recovered to the pre-ischemic level within 1 day after reperfusion. The average increase in the CAP threshold on the seventh day was significantly less in the Ad-GDNF group than in the Ad-LacZ or artificial perilymph group (^*P<0.05, ^**P<0.01, respectively). All values are presented as the means.d. Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.

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Figure 10 shows specimens from the Ad-LacZ vector group 7 days after ischemia stained with rhodamine-phalloidin (Figure 10a) and Hoechst 33342 (Figure 10b). The stereocilia and nuclei of the hair cells disappeared sporadically. In contrast, Figure 11 shows specimens from the Ad-GDNF vector group 7 days after ischemia stained with rhodamine-phalloidin (Figure 11a) and Hoechst 33342 (Figure 11b). The hair cells remained intact in this group.

Figure 10.

Hair cells 7 days after transient ischemia in the Ad-LacZ group. Representative inner and outer hair cells stained with rhodamine-phalloidin (Figure 6a) and Hoechst 33342 (Figure 6b) seen seven days following transient cochlear ischemia/reperfusion in the Ad-LacZ group. Damaged hair cells lacking both stereocilia and nuclei (arrowhead) were observed sporadically. In contrast, all cells were intact in the Ad-LacZ group. Scale bars: 20 m.

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Figure 11.

Hair cells 7 days following transient ischemia in the Ad-GDNF group. Representative inner and outer hair cells stained with rhodamine-phalloidin (Figure 7a) and Hoechst 33342 (Figure 7b) seen 7 days after transient cochlear ischemia/reperfusion in the Ad-GDNF group. All cells were intact in the Ad-LacZ group. Scale bars: 20 m.

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The rates of IHC and OHC damage 1, 4, and 7 days after surgery are summarized in Figure 12 and Figure 13. In the control groups, the mean IHC and OHC loss rates increased gradually until 4 days after reperfusion and remained constant thereafter. Cell loss was more prominent in IHCs than in OHCs. On the seventh day, the mean percentage losses of IHCs and OHCs were 17.04.0 and 3.13.1% in the Ad-LacZ group, and 16.44.8 and 2.92.4% in the artificial perilymph group, respectively. In the Ad-GDNF group, the mean percentage losses of IHCs and OHCs 7 days after ischemia were 4.61.7 and 2.42.3%, respectively. The mean percentage loss of IHCs in the Ad-GDNF group was significantly smaller than in the control groups (P<0.01).

Figure 12.

Rates of IHC loss following transient cochlear ischemia/reperfusion. The rates of inner hair cell (IHC) loss 1, 4, and 7 days after transient cochlear ischemia–reperfusion. In the Ad-LacZ and artificial perilymph groups, the mean IHC loss rates increased gradually until 4 days after reperfusion and remained constant thereafter. On the seventh day, the rates of inner and outer hair cell loss in the Ad-GDNF group were significantly smaller than those in both the Ad-LacZ and artificial perilymph groups (^*P<0.05, ^*P<0.01, respectively). All values are presented as the means.d. Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.

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Figure 13.

Rates of OHC loss following transient cochlear ischemia/reperfusion. The rates of outer hair cell (OHC) loss 1, 4, and 7 days following transient cochlear ischemia/reperfusion. There were no statistical differences among the three groups. All values are presented as the means.d. (n=6 for each group). Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.

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Severity of the ischemic insult

Finally, to eliminate the possibility that the severity of the ischemic insult varied among the three groups, we measured the rates of damage to IHCs and OHCs in the nontreated cochlea from the same animals. As shown in Figure 14, there were no differences among the three groups. These data indicated that the severity of ischemic insult was essentially the same among the three groups.

Figure 14.

Rates of inner and outer hair cell loss on the nontreated side. The rates of inner and outer hair cell loss 7 days after transient cochlear ischemia/reperfusion on the nontreated side. There were no statistical differences in the rates of IHC and OHC loss among the three groups. These data indicated that the severity of ischemic insult was roughly the same in all three groups. All values are presented as the means.d. (n=6 for each group). Statistical analysis consisted of two-way ANOVA followed by Dunnett's multiple comparison test.

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Discussion

In this study, we showed that adenovirus-mediated overexpression of GDNF completely prevented the hearing loss and progressive IHC loss caused by ischemia–reperfusion injury in the gerbil. Our procedure makes it possible to investigate the inner ear damage that is predominantly observed in IHCs. This is the first report to show the protective effect of adenovirus-mediated GDNF overexpression on IHC degeneration after ischemia and demonstrates the feasibility of gene therapy for sudden hearing loss.

GDNF is a member of the transforming growth-factor beta superfamily. GDNF increases the survival of midbrain dopaminergic neurons in vitro¹⁴ and also has a potent neuroprotective effect on a variety of ischemic neuronal damage in vitro and in vivo. Previously, amelioration of ischemic brain injury when GDNF was applied after middle cerebral artery occlusion was demonstrated in a rodent model.^15,16,17 Moreover, the neuroprotective activity of adenovirus-mediated GDNF gene transfer, which is minimally invasive and maintains hGDNF protein production, has also been reported in focal cerebral ischemia,^18,19 global ischemic brain damage,²⁰ and spinal cord ischemic injury.²¹ In the adult rat ear, GDNF mRNA has been detected using in situ hybridization²² and RT-PCR,²³ and immunohistochemistry has been used to localize GDNF protein²⁴ in the organ of Corti. Recent studies have reported a broader range of targets, including cochlear hair cells. The application of GDNF into the cochlea with a mini-osmotic pump enhanced the protection of hair cells.²²

Intracochlear gene transfer is also being developed as a practical tool for inner ear disease therapy. Previous studies demonstrated that adenovirus-mediated overexpression of GDNF in the cochlea protected against the hearing disturbance induced by acoustic overstimulation¹¹ and ototoxic agents,¹² although the pathological findings in those studies suggested that the effect of GDNF overexpression in countering hearing disturbance was owing to protection of OHCs, which are more easily injured by acoustic overstimulation or ototoxic agents than IHCs.^25,26 However, OHCs appear to act as mechanical input to the IHCs, and IHCs are thought to play a crucial role in information transfer to the central nervous system.²⁷ Our results indicate that adenovirus-mediated overexpression of GDNF prevents degeneration of IHCs, which are vulnerable to ischemia–reperfusion injury.^5,6,7,28

The protective mechanism of GDNF is not fully understood, but in this study, it clearly protected IHCs rather than OHCs. There are a number of possible mechanisms, for example differences in the number and distribution of GDNF receptors, in the amount of caspase activity, and in the accessibility of hair cells to secreted GDNF. Previous work has demonstrated that GDNF diminishes ischemia-induced free-radical release by inhibiting neuronal free-radical synthesis during ischemia,¹⁶ or by reducing neuronal caspase activity in the damaged brain.¹⁷ It has been proposed that free radicals or caspase reactivity in the ear are responsible for ischemic damage, because antioxidants have a protective effect on the CAP threshold shift induced by cochlear ischemia.^29,30 In addition, recent experimental studies have shown that ischemia–reperfusion injury of the inner ear appears to be restricted to IHCs, which is closely related to glutamate neurotoxicity.^5,6,28 GDNF can also protect against chronic glutamate toxicity, either by promoting motor axon outgrowth³¹ or by regulating glutamate receptor promoter activity.³² Although other mechanisms prevent ischemia-induced progressive IHC degeneration in GDNF overexpression, one of these mechanisms is thought to be glutamate release in the perilymph. In addition, the protective effects of GDNF may include nonspecific effects, such as facilitating vascular proliferation in the inner ear, since GDNF is reported to modulate renal blood vessel formation.³³ Further observation is necessary to reveal the molecular mechanisms underlying the effects of GDNF on the inner ear damage caused by ischemia.

Several authors have described the distribution of transfected cells following inoculation of virus vectors into the inner ear.^9,10 The cochlea is particularly suitable as a target for gene therapy. Inoculation at one site results in the spread of the vector throughout the cochlear fluid. The bony capsule prevents the spread of the vector to adjacent tissues, and the immune response may be milder than in other tissues.^23,34 In our study, X-gal staining of the gerbil cochlea revealed transfected cells throughout the cochlea, with the most conspicuous blue-stained cells located in the mesothelial cell layer of the organ of Corti, Reissner membrane, spinal ganglion cells, and around hair cells. Furthermore, using a Western blot technique, we were able to quantify the expression of GDNF protein following Ad-LacZ vector inoculation. To the best of our knowledge, this is the first report that Ad-GDNF vector induces GDNF protein production in the cochlea. These cells are presumed to synthesize excess GDNF protein, which is then secreted into the perilymph to rescue the damaged hair cells.

In conclusion, our study showed that adenovirus-mediated overexpression of GDNF extensively prevents hearing loss and progressive hair cell loss in the gerbil cochlea after transient ischemia compared to control groups. It has been widely postulated that interruption of the cochlear blood flow causes sudden sensorineural hearing loss.^1,2,3 The introduction of an adenovirus inoculation technique for ear treatment may prove very beneficial for saving useful hearing after sudden deafness. In clinical use, however, the Ad-GDNF treatment would be given after the onset of sudden deafness. We are planning to examine the effect of Ad-GDNF when this transfer is performed after ischemic insult. It is also necessary to design a clinical method of cochlear inoculation, whether via the round window membrane or through cochleostomy.

Materials and methods

GDNF adenovirus

A replication-defective recombinant adenovirus carrying human GDNF cDNA (AxCAhGDNF) was constructed according to Miyake et al³⁵ with minor modifications. Briefly, human GDNF cDNA derived from cultured human fetal astrocytes was subcloned into a cassette cosmid pAxCAwt carrying an adenovirus type-5 genome lacking the E3, E1A, and E1B regions to prevent virus replication.^35,36 Cosmid pAxCAwt contains a Swa I cloning site flanked by a cytomegalovirus-enhancer-chicken -actin hybrid (CAG) promoter at the 5' end, and a rabbit globin poly(A) sequence at the 3' end. The cosmid was cotransfected into 293 cells with the appropriately cleaved adenovirus genome lacking the E3 region.³⁷ Recombinant adenovirus was propagated and isolated from the 293 cells, and purified by two rounds of CsCl centrifugation. Generation of recombinant adenovirus containing the bacterial -galactosidase gene (AxCALacZ) has been described previously.³⁷

Animals

These experiments were conducted in accordance with the Guidelines for Animal Experimentation of Ehime University School of Medicine. Adult male Mongolian gerbils weighing 60–80 g were used. The animals were divided into three groups: animals were inoculated with Ad-GDNF vector (Ad-GDNF group) or Ad-LacZ vector (Ad-LacZ group), or perfused with artificial perilymph (artificial perilymph group). The animals were anesthetized with a mixture of 3% halothane and nitrous oxide/oxygen (7:3) gas, and were then maintained with 1% halothane gas. In this study, viral inoculation was done 4 days before ischemia and the effects of Ad-GDNF vector were detected 7 days after ischemia.

Viral inoculation

We injected virus solution into the right cochlea. After anesthesia, the right otic bulla of each gerbil was exposed through a retroauricular incision that revealed the round window. A tube 0.1 mm in diameter (Eicom, Tokyo, Japan) was inserted through the round window with a micromanipulator. The tube was perfused with an isotonic adenoviral suspension (10¹⁰ adenoviral particles per milliliter in Ringer's solution) or artificial perilymph (composition: 137 mM NaCl; 5 mM KCl; 2 mM CaCl₂; 1 mM MgCl₂; 1 mM NaHCO₃; 11 mM glucose, pH 7.4)³⁸ at a flow rate of 0.5 l/min for 4 min using a micro-infusion pump (BRC, Tokyo, Japan).

Ischemia

Anesthetized animals were artificially ventilated via a ventilation tube inserted through the mouth. The tidal volume was set to 1 ml and the rate was set to 70 breaths per minute. The vertebral arteries were exposed bilaterally and dissected free from the surrounding connective tissue via a ventral midline incision of the neck.⁴ Then, 4-0 silk sutures were loosely looped around each artery. Ischemia was induced in both cochleae by applying 5-g weights to the sutures. After 15 min of ischemia, the sutures were removed to allow re-circulation, which was confirmed by visual observation through an operating microscope. Rectal temperature was kept at 37°C by warming the body with a heating lamp during the experimental procedure. After resuscitation, the animals were returned to their cages in an animal center, where the room temperature was maintained at 222°C.

X-gal staining

The transgene expression in cochleae inoculated with the Ad-LacZ vector was confirmed by X-gal staining. Following deep anesthesia, the Ad-LacZ-inoculated side of the otic bulla was immediately removed and the cochlea was perfused intrascalarly with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4, and postfixed for 2 h at 4°C with the same fixative.

For whole-mount staining, cochleae were incubated overnight at 37°C with 100 mg/ml X-gal in dimethylformamide, containing 0.5 M K₃Fe(CN)₆ and 0.5 M K₄Fe(CN)₆. After rinsing in PBS, the lateral walls of the cochleae were removed and observed under stereoscopic magnification. A surface preparation technique was then used to remove the otic capsule, lateral wall and tectorial membrane and the bony modiolus with the organ of Corti was carefully detached at the base of the cochlea.

For cryostat-section staining, cochleae were decalcified for 3 weeks in EDTA with paraformaldehyde and processed for cryosectioning embedding. Sections were cut at 10 m and mounted on poly-L-lysine-coated slides and stained overnight at 37°C with 100 mg/ml X-gal in dimethylformamide, containing 0.5 M K₃Fe(CN)₆ and 0.5 M K₄Fe(CN)₆.

Western blot analysis

Following deep anesthesia with an intraperitoneal injection of pentobarbital sodium (50 mg/ml at 0.1 ml/animal), the right otic bulla (wet weight 10 mg) was removed and immediately disrupted and homogenized in an Eppendorf tube containing 100 l of lysis buffer (0.5% SDS, 0.5% Triton-X, 100 M phenylmethane sulfonyl fluoride, 20 mM Tris-HCl pH 8.0). The homogenates were sonicated on ice and centrifuged at 13 000 rpm for 10 min at 4°C. The protein content in the supernatant was determined using a BCA protein assay kit (Pierce, Rockland, IL, USA) with bovine serum albumin as a standard.³⁹ The supernatant was mixed with sample buffer (62.5 mM Tris-HCL, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol and 0.001% bromophenol blue) to a final protein concentration of 1 mg/ml. The samples were boiled for 5 min. Equal amounts of protein (15 g/lane) were resolved by SDS-PAGE electrophoresis, transferred onto nitrocellulose membrane, and immunoblotted with affinity-purified rabbit polyclonal anti-GDNF antibody (Santa Cruz, Santa Cruz, CA, USA) using previously described procedures.⁴⁰

To quantify the GDNF levels, densitometric analysis of scanned bands was performed. The integrated optical density was obtained from digitized Western blot data using the program NIH Image. The data were normalized to internal standards (no-treatment animals) on each gel and given as percentage values. Statistical analysis was performed by one-way ANOVA followed by Dunnett's multiple comparison test.

Monitoring hearing function

To evaluate virus toxicity, the change in hearing function after injection of Ad-GDNF vector, Ad-LacZ vector, or artificial perilymph was measured in 18 gerbils (n=6 for each group) by recording the CAP threshold shift before and 4, 8, and 11 days after injection. In addition, the change in hearing function after injection of Ad-GDNF vector, Ad-LacZ vector, or artificial perilymph was measured in 18 gerbils (n=6 for each group) by recording the CAP threshold shift before and 1, 4, and 7 days after ischemia. CAPs were recorded using the method of Hildesheimer et al⁴¹ with minor modification. Animals were anesthetized with a mixture of 1% halothane and nitrous oxide/oxygen (7:3). Following exposure of the right otic bulla, a platinum reference electrode coated with epoxy resin was placed into the facial nerve canal via the stylomastoid foramen. It was fixed to the bony bulla with dental cement, fed under the scalp, and led outside the skin at the vertex. An indifferent electrode made of a stainless-steel needle was placed in the ipsilateral mastoid muscle during measurement.

As the animals could not tolerate long anesthesia, the CAP in response to an 8000-Hz tone burst (0.5 ms rise/fall time and 10 ms duration) was measured using a signal processor (NEC Synax 1200, NEC Medical Systems, Japan). This frequency was selected since the higher frequency region of the cochlea was found to be more vulnerable to ischemic injury than the middle or lower frequency regions in our previous study.⁷ The responses were processed through a 50-Hz to 3-kHz band-pass filter and averaged 300 times. The sound pressure in front of the tympanic membrane was monitored using a small microphone incorporated in to the conduction tube of a sound stimulator. The CAP threshold was determined by applying acoustic stimuli in 10-dB steps, except near the threshold, where they were applied in 5-dB steps.

Tissue preparation

Hair cell loss was identified by staining specimens with both rhodamine-phalloidin and Hoechst 33342. The former is better for observing the stereocilia of hair cells and the latter is better for evaluating their nuclei.

Animals were killed 1, 4, or 7 days after ischemia (n=6 for each group). Following deep anesthesia, both otic bullae were immediately removed and the cochleae were perfused intrascalarly with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4, and postfixed for 2 h at 4°C with the same fixative. The cochleae were then immersed in phosphate-buffered saline (PBS) at pH 7.4, and the organ of Corti was dissected. Specimens were then stained for 30 min at room temperature with rhodamine-phalloidin (Molecular Probes, Eugene, OR, USA), which was diluted 250 times in PBS containing 0.25% Triton-X-100 and 1% bovine serum albumin. After rinsing in PBS, the cochleae were stained with Hoechst 33342 (20 g/ml; Calbiochem-Novabiochem Corporation, La Jolla, CA, USA) prepared in PBS in a dark room for one hour. They were then rinsed again in PBS and mounted in carbonate-buffered glycerol (one part 0.5 M carbonate buffer, pH 9.5: nine parts glycerol) containing 2.5% 1,4-diazabicyclo-(2,2,2)-octane to retard bleaching of the fluorescent signal. Fluorescence was detected using an Olympus BX60 microscope with green (BP 546, FT 580, LP 590) and UV (BP 365, FT 395, LP 397) filters. The numbers of intact and dead hair cells in the basal turn of the cochlea were counted, and the ratio of intact to dead hair cells was calculated.

The statistical significance of differences in the average increase of the CAP threshold and the rate of hair cell loss between the Ad-GDNF or Ad-LacZ groups and the artificial perilymph group were evaluated by ANOVA followed by Dunnett's multiple comparison test.

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