The apoptosis of cochlear cells, including hair cells and spiral ganglion cells, is the final common pathway for many hearing disorders including drug-induced hearing loss.1,2 Bcl-2, a key regulator of apoptosis, promotes cell survival by inhibiting adapters needed for activation of the proteases (caspases) that dismantle the cell.3,4 Gonzalez-Garcia and colleagues5 have cloned and characterized the murine Bcl-x gene, whose human counterpart displays striking homology to Bcl-2 and prevents cell death by a similar mechanism as Bcl-2. Bcl-xL is the major Bcl-x mRNA form expressed during murine development. Recent studies have demonstrated that Bcl-xL inhibited neurodegeneration in progressive neurological disorders and protected the heart against ischemia/reperfusion injury.6,7 Studies over the last decade have left little doubt that aminoglycosides induce apotosis of the cochlear cells by producing reactive oxygen species (ROS), including free radicals, in the mitochondria.1,2 Bcl-xL is an integral membrane protein localized primarily in the mitochondrial membrane and nuclear envelope.5 It suppresses cell death presumably by preventing the release of apoptogenic factors from the mitochondria and directly interacting with caspases.8,9 Thus, Bcl-xL appears to be a robust endogenous cellular survival factor and an attractive target for molecular therapeutic intervention in aminoglycoside ototoxicity.
We have efficiently transduced cochlear cells with serotype 2 of adeno-asociated virus (AAV2) in our previous study.10 Using this method, we evaluated the protective effect of AAV2-mediated transgene expression of Bcl-xL as a potential therapeutic agent in a murine model of aminoglycoside-induced hearing impairment in the present study.
Construction and preparation of the proviral plasmids
pAAV2-EGFP is an AAV vector proviral plasmid with enhanced humanized green fluorescent protein (EGFP), which is driven by the chicken β-actin promoter with cytomegalovirus enhancer unit (CAG) promoter, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and AAV2 inverted terminal repeats (ITRs).10 Plasmid pAAV2-Bcl-xLflag harbors the mouse Bcl-xL cDNA tagged with a FLAG sequence (DYKDDDDK) at the N-terminal expression cassette under control of CAG, human growth hormone first intron, and SV40 early polyadenylation sequence flanked by ITRs. A schematic illustration of the AAV vectors was shown (Fig. 1). AAV-helper plasmid harbors Rep and Cap. Adenovirus helper plasmid pAdeno5 is identical to pVAE2AE4-5 and it encodes the entire E2A, E4 regions and VA RNA I and II genes.11 Plasmids were purified with the QIAGEN plasmid purification Kits (USA).
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Fig. 1. A: Schematic representations of viral vectors constructed. AAV vector was constructed using CAG to drive mouse Bcl-xL with flag and polyA. ITR: inverted terminal repeats from AAV2. B: Western blot analysis of cell extracts from HEK293 cells after transduction with AAV2-Bcl-xL. HEK293 cells transduced with mock or AAV2-EGFP was used as negative controls. Expression of Bcl-xL was detected by anti-FLAG antibody.
Recombinant AAV vectors production
AAV2 vectors were produced based on the three-plasmid transfection by an adenovirus-free system, as previously reported.11 AAV2 vectors were produced with the AAV packaging plasmids pHLP19, proviral plasmid pAAV2-Bcl-xLflag or pAAV2-EGFP, respectively. Production of vectors used in cell culture experiments was performed. The cells were co-transfected with 650 µg of each following plasmid: AAV proviral plasmid pAAV2-Bcl-xLflag, AAV-helper plasmid, and adenovirus helper plasmid pAdeno5, by a calcium phosphate co-precipitation method. Recombinant AAV2 was harvested at 72 hours after transfection by three cycles of freeze/thawing. The vector lysate was then purified twice on a cesium chloride (CsCl) gradient. The physical titers of virus stock were determined by quantitative real-time PCR of DNase-treated stocks.12
In vitro expression of AAV2-Bcl-xLflag
The Bcl-xL protein expression was confirmed by Western blotting. To detect the in vitro expression of Bcl-xLflag fusion protein, 293 cells were transduced with AAV2-Bcl-xLflag or AAV2-EGFP (1×104 vector genome copies (g.c.)/cell). Forty-eight hours after transduction, cell lysates were separated by 12% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Residual protein-bindings sites were blocked with 5% skim milk in TBS for 1 hour at room temperature. Membranes were incubated overnight at 4˚C using mouse anti-FLAG M2 primary antibody (1:1000, Sigma, St Louis, MO, USA), followed by incubation with a secondary anti-mouse conjugated to horseradish peroxidase (1: 2500, Amersham, Arlington Heights, IL, USA) for 1 hour at room temperature. Chemiluminescent signals were detected by the ECL systems (Amersham Biosciences UK Limited, Buckinghamshire, England).
Surgical procedures and cochlear perfusions
All animal studies followed the guidelines issued by the Committee of Animal Research at Peking University. After the mice were anesthetized, a postauricular approach was used to expose the tympanic bony bulla. A small opening (2 mm) to the tympanic bulla was carefully drilled through the bone of the bulla to provide access to the round window membrane. Five µl AAV2 vectors solution (1×109 g.c.) was microinjected into the cochlea through the round window over 10 minutes via a glass micropipette (40 μm in diameter) which was connected to a UNIVENTOR 801 syringe pump (High Precision Instruments, UNIVENTOR Ltd., Malta). Artificial perilymph (AP) (145 mmol/L NaCl, 2.7 mmol/L KCl, 2 mmol/L MgSO4, 1.2 mmol/L CaCl2, 5 mmol/L HEPES) was used as the control. Twenty-five healthy adults (4 weeks-old) female C57BL/6J mice with normal Preyer's reflexes weighing 12−15 g were used in the present study. The animals were divided into four groups: Vehicle control group: AP administration (n=5); AP/kanamycin group: AP followed by kanamycin administration 5 days later (n=5); AAV2-EGFP/kanamycin group: AAV2-EGFP followed by kanamycin administration 5 days later (n=5); AAV2-Bcl-xL/kanamycin group: AAV2-Bcl-xL followed by kanamycin administration 5 days later (n=10).
Kanamycin sulfate was dissolved in saline at concentration of 80 mg kanamycin base/ml so that a dose of 800 mg of kanamycin base/kg body weight was obtained by injecting 0.01 ml/g body weight. Five days after injection of AAV2 vectors or AP, kanamycin were administrated subcutaneously twice daily for 15 consecutive days. Body weight of animals was monitored daily and the administered drug dosages were adjusted accordingly.
Assessment of cochlear function by auditory steady state response (ASSR)
Auditory thresholds were determined by ASSR audiometry (Tucker-DAVIS Technologies, FL, USA) and Scope for Windows software (Power Lab, ADInstruments Pty Ltd, USA). The lowest stimulus level that yielded a detectable ASSR waveform was defined as the threshold. Thresholds were taken for each animal prior to the beginning of the study, and day 2 after finishing administration of kanamycin. The ASSR was measured as previously described.10 In brief, ASSR was elicited and measured, at 4, 8, 12, 16, 20, and 24 kHz frequency with click tone in systematic 5-dB steps. Around the threshold, responses for 512 sweeps were averaged at each intensity level.
In vivo expression of AAV2-Bcl-xLflag
The exogenous Bcl-xL protein expression was detected by immunohistochemistry with anti-flag antibody. After fixing and decalcifying, the cochleae were made coronal or transverse cryosection (10 µm). Sections were incubated with primary antibodies for Bcl-xLflag (1:200, mouse anti-flag monoclonal antibody), followed by incubation with biotinylated secondary antibodies (to species of primary antibodies) (1:500, Santa Cruz, USA). Sections were visualized with avidin-biotinylated peroxidase complex procedure (Vectastain ABC kits; Vector Laboratories, Burlingame, CA, USA) by using Vector SG as a chromogen (SK-4700, Vector SG Substrate; Vector Laboratories, Burlingame, CA, USA).
Tissue preparation for the cochlear preservation
After decalcification, the whole cochleae were microdissected. The lateral wall, tectorial membrane and Reissner's membrane were removed, and the modiolus with the organ of Corti was detached from the temporal bone. The modiolus with the organ of Corti was immersed in 0.1% Triton-X-100 in PBS for 20 minutes, followed by incubating for 30 minutes at room temperature in 80 nmol/L rhodamine phalloidin (Sigma Chemical Co., MO, USA). The organ of Corti was mounted on glass slides with Crystal/Mount to check the hair cells and was photographed under a fluorescence microscope OLYMPUS IX70 (Olympus Corporation, Tokyo, Japan). The Studio Lite software (Olympus Corporation, Tokyo, Japan) was used to analyze the pictures.
Results are presented as the means ± standard deviation (SD). Data were statistically analyzed using repeated-measures analysis of variance (ANOVA) followed by paired Student's t test (inoculated and contralateral ears) and unpaired Student's t test (inoculation and control groups) (StatView 5.0 software, SAS Institute Inc, NC, USA). P<0.05 was considered statistically significant.
In the present study, ASSR thresholds shift after injection of vector and kanamycin was calculated to evaluate the protective effect of AAV2-mediated Bcl-xL on the cochlea. ASSR thresholds shift had no differences between AP and AAV2-EGFP control group (P>0.05). The ASSR thresholds shift was significantly lower in the AAV2-Bcl-xL group than that in the AAV2-EGFP control group at each frequency tested (P<0.05, Fig. 2). Meanwhile, the AAV2-Bcl-xL group had significantly lower ASSR thresholds shift comparing with the contralateral normal ears at each frequency tested (P<0.05, Fig. 3). AAV2-Bcl-xL group showed a large variability of ASSR threshold shifts at each frequency after kanamycin administration. However, 7 of the 10 mice in the AAV2-Bcl-xL group showed better hearing threshold in the inoculated ears than the uninoculated ears.
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Fig. 2. ASSR threshold shifts of inoculated side (mean ± SD) in AAV2-EGFP and AAV2-Bcl-xL groups. Significant differences between the two groups were detected at different tested frequency (t test; 4 kHz: t=6.822, P=0.0024; 8 kHz: t=5.143, P=0.0036; 12 kHz: t=8.657, P=0.0021; 16 kHz: t=10.621, P=0.0017; 20 kHz: t=12.108, P=0.0003; 24 kHz: t=10.171, P=0.0009).
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Fig. 3. ASSR threshold shift (means ± SD) at different tested frequency in the inoculated (right side) and contralateral ear (left side) of the AAV2-Bcl-xL /kanamycin group at 2 weeks after the inoculation. Significant differences between two sides were detected at each tested frequency (t test; 4 kHz: t=7.269, P=0.0001; 8 kHz: t=12.314, P<0.0001; 12 kHz: t=6.353, P=0.0001; 16 kHz: t=11.114, P<0.0001; 20 kHz: t=6.917, P=0.0002; 24 kHz: t=5.512, P=0.0004).
We also examined the effect of AAV2-mediated transgene Bcl-xL on the augmentation of the survival of the cochlear cells, especially hair cells. In the AAV2-Bcl-xL group, protection of outer hair cells was detected in all inoculated ears comparing with the uninoculated ear. In the inoculated ear, outer hair cells loss in the AAV2-Bcl- xL group was less than that of the AAV2-EGFP group and the AP group (Fig. 4). In sites of missing outer hair cells, typical scars were found (Fig. 4). In most mice, outer hair cells loss increased from apex to base, bilaterally. Thus, exogenous Bcl-xL expression had a protective effect on damaged cochlear cells by inhibiting kanamycin-induced apoptosis. Exogenous Bcl-xL expression in mouse cochlea was detectable by immunohistochemistry with anti-flag antibody (Fig. 5).
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Fig. 4. Cochlear tissue stained with rhodamine phalloidin and observed under fluorescence microscope. A: The normal cochlea. B: The organ of Corti in the AAV2-Bcl-xL/kanamycin group was well protected by Bcl-xL. No loss of outer hair cells was observed in the organ of Corti. C: The organ of Corti in the AAV2-EGFP/kanamycin group. A few outer hair cells are absent, replaced by scars (arrows). Scale bar=25 µm.
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Fig. 5. A: AAV2-Bcl-xL-innoculated cochlea showing strong Bcl-xL expression in the spiral ganglion cells with immunohistochemistry. B: No Bcl-xL expression in the spiral ganglion cells of control cochlea. Scale bar=10 µm.
Enormous experimental evidences have demonstrated that ROS participate in the cellular events which lead to aminoglycoside-induced hearing loss. The over- production of ROS causes membrane lipid peroxidation, initiation of caspase activation, and subsequent cellular apoptosis.13 The pathology of aminoglycoside-induced hearing loss is similar across species, including human being. The primary effect of aminoglycoside in the cochlea is destruction of outer hair cells, which progresses from the base to the apex. Meanwhile, hearing loss advances from high to low frequencies. Emerging evidence has suggested that a significant portion of the cochlear cell death after aminoglycoside ototoxicity is attributable to an active type of cell death reminiscent of apoptosis, in which a number of apoptosis-regulatory gene products are activated.14 The protective effect of Bcl-xL against aminoglycoside-induced hearing impairment may be mediated by several mechanisms. Firstly, Bcl-xL can prevent the release of apoptogenic factors from the mitochondria by directly inhibiting caspase-3 activation in injured cochlear hair cells.5 Furthermore, Bcl-xL could block mitochondrial release of other apoptogenic factors such as apoptosis-inducing factor, which induces mediate cell death by caspase-independent mechanisms.15 Finally, Bcl-xL also protects cell against hypoxia-induced necrosis,16 which is one of the pathological mechanisms of aminoglycoside- induced cochlear cells death.1
Here, we studied the protective effect of AAV2-mediated Bcl-xL as a potential therapeutic agent in a murine model of aminoglycoside-induced hearing loss. The present data showed that the AAV2-Bcl-xL —inoculated ears have better hearing function and less structural damage comparing with the contralateral (uninoculated) ears of the same animals or the control ears of the control groups. The delivery of the Bcl-xL gene into cochlea via round window is now practical. Therefore, AAV2-mediated Bcl-xL may become a potential therapeutic agent to prevent apoptosis in some cochlear degenerative disorders.
Our previous study has demonstrated that AAV vectors can transfer genes of interest to a wide variety of cells with high efficiency in the cochlea in vivo.10 AAV2-mediated transgene can be expressed in tissues containing long-surviving postmitotic cells such as the cochlear ganglion cells and inner hair cells. The transduction of cochlear ganglion cells with AAV2-mediated Bcl-xL gene could prevent these cells from aminoglucoside-induced ototoxicity, it may also protect outer hair cells, which have no Bcl-xL transduction, from death through cell-cell interaction. Basile and colleagues reported that N-methyl-D-aspartate (NMDA) agonists may indirectly protect hair cells from ototoxicity induced by aminoglycosides through interaction with the spiral ganglion cells,17 which may explain the mechanism of Bcl-xL transduction.
We found in the present study that the protecting effect of Bcl-xL gene transduction varies among individual mouse dramatically. The reason for this variation is not clear, but it probably results from several factors, including the efficacy of the infusion in the operation, the amount of AAV2-Bcl-xL, and different sensitivity to the toxins among individuals. It was reported that adenovirus mediated Bcl-xL, gene could promote retinal ganglion cell neurite regeneration.18 The spiral ganglion cell neurite regeneration after AAV2-Bcl-xL transfected cochleae warrants further exploration.
The effectiveness of Bcl-xL in the insulted cochlear preservation was remarkable in our study, although the interpretation for the survival of cochlear apical hair cell and the function preservation remains uncertain. We did not clarify the mechanism of augmentation of the survival. It is hypothesized that the interaction between the spiral ganglion cells and the potential presence of other survival factors in the remaining apical organ of Corti indirectly protects the cochlear apex function. This study suggests that stabilization of the mitochondrial membrane by Bcl-xL over-expression can prevent cochlear cells from apoptosis. Thus, gene therapy with AAV2-mediated Bcl-xL may provide a new therapeutic strategy for diseases characterized by pathological forms of apoptosis.
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