American Journal of Otolaryngology - Head and Neck Medicine and Surgery
Volume 32, Issue 1 , Pages 8-12, January 2011

Study of protective effect on rat cochlear spiral ganglion after blast exposure by adenovirus-mediated human β-nerve growth factor gene

  • Jian Wu, MD

      Affiliations

    • Department of Otorhinolaryngology Head and Neck Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China
    • Corresponding Author InformationCorresponding author. Department of Otorhinolaryngology Head and Neck Surgery, Shanghai Changzheng Hospital, Second Military Medical University, 415 FengYang Road, Shanghai, 200003 China. Tel.: +86 021 81885962.
    • ,
  • Bing Liu, MD

      Affiliations

    • Department of Otorhinolaryngology Head and Neck Surgery, the Affiliated Hospital of Jining Medical College, Jining, Shangdong Province, China
    • ,
  • Jingping Fan, MD

      Affiliations

    • Department of Otorhinolaryngology Head and Neck Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China
    • ,
  • Qiubei Zhu, MD

      Affiliations

    • Department of Otorhinolaryngology Head and Neck Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China
    • ,
  • Jiang Wu, MD

      Affiliations

    • Department of Otorhinolaryngology Head and Neck Surgery, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China

Received 15 June 2009 published online 21 December 2009.

Article Outline

Abstract 

Objective

To study whether adenovirus-mediated human β-nerve growth factor (Ad-hNGFβ) gene has any protective effect on rat cochlear spiral ganglion after blast exposure.

Methods

Deafness was induced by blast exposure (172.0 dB) in 20 healthy rats. Seven days after blast exposure, Ad-hNGFβ was infused into the perilymphatic space of 10 animals as the hNGFβ/blast group, and artificial perilymph fluid (APF) was infused into the perilymphatic space of 10 animals as the APF/blast control group. An additional control group consisted of 10 healthy rats which received Ad-hNGFβ target gene with no blast exposure (hNGFβ/control group). Auditory functions were monitored by thresholds of auditory brain stem responses (ABR). At weeks 1, 4, and 8 postoperatively, the animals were killed, and the cochleae were removed for immunohistochemical, hematoxylin and eosin staining study.

Results

The ABR threshold shifts in the hNGFβ/blast group were significantly smaller than that of APF/blast control group. There were no significant differences of the ABR values between before and after operation in the hNGFβ/control group. Expression of Ad-hNGFβ protein was detected in each turn of the cochlea in the first week, with almost equal intensity in all turns. In the fourth week, the reactive intensity decreased. In the eighth week, no reaction was detectable. The results of hematoxylin and eosin stain showed that the number of spiral ganglions in the hNGFβ/blast group was significantly greater than that of the APF/blast control group in the 4th week (P < .01).

Conclusion

Adenovirus-mediated human β-nerve growth factor can be expressed at a high level and for a relatively long period in the blast impaired cochlea, suggesting that Ad-hNGFβ has a protective effect on rat cochlear spiral ganglion cells after blast exposure.

 

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1. Introduction 

The advent of recombinant DNA technique stimulates the basic research and attempted use of human gene therapy for the prevention and treatment of sensorineural deafness. Delivering gene products into the inner ear has been shown to afford a significant degree of protection and rescue against acoustic trauma; some researchers attempted to use recombinant BDNF (brain-derived neurotropic factor) gene in the prevention and treatment of steady noise hearing impairment [1], [2], [3], [4], [5], [6]. Our previous experiment preliminarily demonstrated that human nerve growth factor (hNGF) had a preventive effect on blast hearing impairment [7]. The goal of the present study which we use adenovirus-mediated human β-nerve growth factor gene (hNGFβ) in a rat blast deafness model is to demonstrate whether hNGF gene has any protective effect on blast hearing impairment. This kind of study has not been reported in any previous studies.

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2. Materials and methods 

2.1. Experimental rats 

Animals were obtained from the experimental animal center of Second Military Medical University (Shanghai, China). All animal experiments were approved by Second Military Medical University and were performed using accepted veterinary standards. Thirty healthy white adult rats weighing 200–210 g with normal Preyer's reflex were used in the present experiment. The blast source came from a specially designed D-86 spark pulse generator (Tongji University, Shanghai, China). The animals were anesthetized with 30 mg/kg IP barbital and fixed around a circle of 10 cm in diameter on a special wood board with the head placed centripetally. The frequency spectrum and intensity of the blast in the blast area were monitored with a Bruel & Kjaer 2230 precise sound-level meter, ensuring that the peak was 172.0-dB sound pressure level (SPL). Twenty rats were deafened, and them be exposed to the blast source 30 times with 2-second intervals and 0.5-millisecond pulse width. Auditory brainstem response (ABR) testing was used to assess hearing thresholds in normal animals and to verify effectiveness of deafening. Seven days after blast exposure, ABR testing was done to establish criteria for inclusion of the study (thresholds shifts > 75dB SPL). Deafened rats were assigned randomly to 2 groups, 10 rats in the study group (Ad-hNGFβ target gene was transferred into left inner ears, hNGFβ/blast group), 10 rats as the control group (artificial perilymph fluid [APF] was transferred into left inner ears, APF/blast control group). An additional control group consisted of 10 healthy white adult rats which received Ad-hNGFβ target gene with no blast exposure (hNGFβ/control group).

2.2. Method of gene transfer 

The experimental rats were anesthetized with 30 mg/kg IP barbital and spread with sterile drapes routinely. The skin and muscle tissue were sheared off from the left posterior ear to expose the otic vesicle, which was cut open from the pars dorsalis to expose the basement turn of the cochlea. A 1-mm hole near the fenestra cochleare was drilled by using a fine needle under microscope, through the small hole a 10-μL microinjector was inserted into the perilymph fluid and Ad-hNGFβ (Institute of Clinical Medicine Yunyang School of Medicine, Yunyang, China) was injected slowly, 10 μL per ear totaling 1 × 109 viruses for the hNGFβ group, the hNGFβ/blast group and the hNGFβ/control group, and 10-μL APF for the APF/blast control group.

2.3. Sampling of internal ear specimens 

The rats were decapitated at first, fourth and eighth weeks postoperatively; the otic vesicle was removed and opened to expose the cochlea. A small hole was drilled from the top of the cochlea, into which 4% paraformaldehyde was injected to fix and emerge the cochlea at 4°C overnight. The specimen was then decalcified with 10% EDTA for 7 days, washed with distilled water, dehydrated, hyalinized, paraffin-embedded, sectioned, and then immunohistochemically stained with hNGFβ antibody. Spiral ganglion cells were stained with hematoxylin and eosin staining.

2.4. Detection of hNGFβ expression 

The hNGFβ positive reaction product in the cochlea was detected by immunohistochemical EnVision method. The primary antibody we used is rabbit anti human hNGFβ (Wuhan Boster Biological Technology Co, Ltd, Wuhan, China). The specimen was diaminobenzidine-colorized and resin-mounted for light microscopic observation.

2.5. Microscopic counting of spiral ganglion cells 

Slides for counting were chosen randomly among those quality morphology. In each canal, only the number in the first and second turn was counted. The spiral ganglion cells were quantitated using the MetaMorph Imaging System (Universal Imaging Corp, West Chester, PA). The computer mouse was used to click over every spiral ganglion cell that contained a nucleus. Once counted, cells were labeled so they could not be counted twice. Four weeks after operation, the number of the spiral ganglion cells of the cochlea was selected for quantitation.

2.6. Statistic analysis 

The data was analyzed using Sigma Stat statistical software (SPSS/Jandel Scientif Software, Chicago, IL). The average cell density values of each group were used by 2-sample t test to determine the degree of statistical difference. In all cases, P < .01 was considered statistically significant.

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3. Results 

A daily postoperative observation revealed a complete recovery of the animals, with no head tilt, no loss of appetite, and no other signs of ear specific or systemic toxicity in any rats. There was no intracochlear infection.

3.1. Measurement of ABR threshold 

Auditory brainstem response tests were performed on deafened animals to ascertain that the deafening procedure was successful. There were no significant differences of the click-ABR thresholds in rats among all animals before blast exposure. However, there were significant differences between the thresholds recovering in the hNGFβ/blast group and that of the APF/blast control group (Table 1); the threshold shifts in the hNGFβ/blast group were significantly smaller than that of the APF/blast control group. There were no significant differences between these values before and after operation in the same rats in the hNGFβ/control group (before operation: 25.6 ± 3.02 dB SPL, after operation: 26.1 ± 3.38 dB SPL).

Table 1. ABR threshold at various time in the 2 groups of rats ()
Group 4 weekPre-exposureProexposurePostoperative 1 wk4 wk
hNGFβ/blast group25.5 ± 3.4178.3 ± 5.1646.4 ± 5.1326.7 ± 3.62
APF/blast group25.1 ± 3.3277.4 ± 5.2460.5 ± 5.2557.6 ± 4.76

The ABR threshold shifts at week 1, 4 after gene transfer in the hNGFβ/blast group rats were significantly smaller than those in the APF/blast control group (P < .01).

3.2. Ad-hNGFβ expression in the cochlea of rats 

Extensive expression of hNGFβ protein was found in the cochlea in the 1st week of Ad-hNGFβ transfer. Staining was seen from the basement turn to the top turn, with almost equal stain intensity. The main expression was found in supporter cells, spiral ganglion, spiral limbus, and spiral ligament of the basement membrane area (Fig. 1). In the fourth week of gene transfer, hNGFβ protein expression was still detectable, and the stain intensity in the basement membrane area decreased compared with that in the first week (Fig. 2). In the eighth week of gene transfer, immunohistochemical reaction was negative, and no expression of hNGFβ protein was detected. Immunohistochemical reaction in all animals of the control group was negative.

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  • Fig. 1. 

    Immunohistochemical result 1 week after Ad-hNGFβ transfer. Staining was seen from the basement turn to the top turn, with almost equal intensity, the main expression was found in supporter cells, spiral ganglion, spiral limbus, and spiral ligament of the basement membrane area (original magnification ×100).

  • View full-size image.
  • Fig. 2. 

    Immunohistochemical result 4 weeks after Ad-hNGFβ transfer. hNGFβ Protein expression was still detectable; the stain intensity in the basement membrane area decreased as compared with that in the 1st week (original magnification ×100).

3.3. Results of spiral ganglion cell counting 

Hematoxylin and eosin–stained sections showed that the mean number of spiral ganglion cells in the cochlea of hNGFβ/blast group was significantly greater than that of the APF/blast control group (145.6 + 2.60 vs 82.4 + 3.25, P < .01), and the configuration of these cells was similar to that of normal animals (Fig. 3A). In the APF/blast control group, large areas of spiral ganglion cells were found to be necrotic (Fig. 3B).

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  • Fig. 3. 

    Hematoxylin and eosin staining. A, The mean number of spiral ganglion cells in the cochlea of hNGFβ/blast group was significantly greater than that of the APF/blast control group in the fourth week gene transfer, the configuration of these cells was similar to that of normal rats. B, In the APF/blast control group, large areas of spiral ganglion cells were found to be necrotic (original magnification ×400).

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4. Discussion 

Dramatic advances in gene engineering technology in recent years have made it possible to use gene therapy in the treatment of internal ear disorders. The cochlea has several advantages as a target for gene transfer. The cochlea anatomically contained bony capsule, which provides isolation from surrounding tissues. A vehicle transferred from any point site may distribute swiftly throughout the cochlea via the perilymph fluid, without likelihood of diffusing to the adjacent tissues. It is possible to quantify spiral ganglion cells [8], [9], [10], [11], [12] and to take sensitive physiological tests; these further increase the value of the cochlea as a target organ for gene transfer, also it's safe to transfer gene into the rat cochlea [13], [14], [15].

Neurotrophic factors can influence neuronal development, growth, and survival [16]. Among the known neurotrophic factors is the family of neurotrophins, which includes nerve growth factor (NGF), BDNF, NT-3, and NT-4/5. It has established several experimental models that growth factors have protection and rescue effect in the inner ear [1], [2], [5]. Nerve growth factor is the earliest identified growth factor, consisting of three subunit uncovalent bonds: α, β, and r, of which β is the only subunit that possesses NGF activities. β Subunit consists of 2 exact same peptide chains, and each chain contains 118 amino acid residues. The amino acids and cDNA of β subunit have been defined. It is generally accepted that exogenous NGF could promote repair and re-generation of impaired nerves, regulate cell proliferation and differentiation, and maintain cell viability. Cochlear hair cell is able to synthesize and secrete series factors of the NGF family, and nourish spiral ganglion cells. Blast cause damage to hair cells so that spiral ganglion cells lose support and protection of various NGFs, resulting in neural degeneration.

Recombinant adenovirus is a vehicle extensively used in the research of gene therapy [17], [18], [19]. The present study showed that Ad-hNGFβ has high-titer virus and high transfer efficiency. The observations of our experiment also showed that 1 week after transferring hNGFβ, there was extensive expression of hNGFβ protein in the cochlea from the basement turn to the top turn, with almost equal stain intensity, and that single-point transfer enabled protein expression throughout the cochlea, indicating successful transfection of Ad-hNGFβ gene transferred. The transfected tissues included support cell in the basement membrane area, spiral ganglion, spiral limbus, and spiral ligament. In the fourth week of transfer hNGFβ protein expression was still detectable, and the stain intensity in the basement membrane area decreased compared with that in the first week, indicating that Ad-hNGFβ may express in the cochlea for a relatively long period. In the eighth week of transfer, no expression of hNGFβ protein was detected, indicating that with the lapse of time the product of exogenous NGF gene expression reduced gradually. As half-life of NGF is short, in vivo drug administration has to be repeated in order to maintain the effective concentration in the cochlea. But each drug infusion would create inconvenience for the next administration. To solve this problem, we used only one gene transfer to maintain a relatively long period of expression, indicating that gene transfection can be maintained steadily for a long period. But the fact that no expression of hNGFβ protein was detected in the eighth week of transfer indicates that the expression of transferred gene is not permanent.

Before blast exposure, the click-ABR thresholds were similar in all animal groups, which indicated that the experimental condition was equal. There were no significant differences between these values before and after transfection in the same rats in hNGFβ/control group, which indicated that Ad-hNGFβ transfection into cochlea had no obvious effects on auditory function of the normal rat, which was consistent with previous studies [20], [21], [22], [23]. However, there were significant differences between the thresholds recovering in the hNGFβ/blast group and that of the APF/blast control group; the threshold shifts in the hNGFβ/blast group were significantly smaller than that of the APF/blast control group, which indicated that the protective effects on auditory neurons by Ad-hNGFβ against blast exposure.

As strong pulse blast causes extremely large damage to cochlear hair cells and spiral ganglion cells of rats, death of hair cells in turn aggravates degeneration of spiral ganglion cells. The present experiment showed that the number of spiral ganglion cells of hNGFβ group was significantly greater than that of the control group, with cell configuration similar to that of normal cells, indicating that Ad-hNGFβ could control degeneration of spiral ganglion cells effectively after blast-induced death of hair cells. The mechanism of protection afforded by Ad-hNGFβ against blast exposure is unclear. We speculate that the mechanisms of protection of Ad-hNGFβ may lie as follow. On one hand, hNGFβ reduces intracochlear damage from oxygen free radicals. Blast reduces blood flow and causes hypoxia in the cochlea, so that enzyme activity in clearing free radicals decreased, and activity and concentration of the antioxidant in the cochlea also decreased, resulting in accumulation of large amounts of free radicals. hNGFβ could raise intracellular antioxidase activity, thus providing a protective effect on blast hearing impairment. On the other hand, hNGFβ prevents cell apoptosis by inhibiting activation of apoptosis-related protease (cysteine).

In conclusion, the data we present demonstrate that Ad-hNGFβ effectively assures viability of sufficient spiral ganglion cells in the rat cochlea after blast exposure, thus exerting a protective effect on blast hearing impairment. This finding has not been reported in previous studies. It creates a favorable condition of effective gene therapy for human inner ear [24], [25].

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PII: S0196-0709(09)00167-7

doi:10.1016/j.amjoto.2009.08.012

American Journal of Otolaryngology - Head and Neck Medicine and Surgery
Volume 32, Issue 1 , Pages 8-12, January 2011

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