Abstract
Objective
To study whether adenovirus-mediated human β -nerve growth factor (Ad-hNGF β ) 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-hNGF β was infused into the perilymphatic space of 10 animals as the hNGF β /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-hNGF β target gene with no blast exposure (hNGF β /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 hNGF β /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 hNGF β /control group. Expression of Ad-hNGF β 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 hNGF β /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-hNGF β has a protective effect on rat cochlear spiral ganglion cells after blast exposure.
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 . Our previous experiment preliminarily demonstrated that human nerve growth factor (hNGF) had a preventive effect on blast hearing impairment . The goal of the present study which we use adenovirus-mediated human β -nerve growth factor gene (hNGF β ) 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.
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-hNGF β target gene was transferred into left inner ears, hNGF β /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-hNGF β target gene with no blast exposure (hNGF β /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- μ L microinjector was inserted into the perilymph fluid and Ad-hNGF β (Institute of Clinical Medicine Yunyang School of Medicine, Yunyang, China) was injected slowly, 10 μ L per ear totaling 1 × 10 9 viruses for the hNGF β group, the hNGF β /blast group and the hNGF β /control group, and 10- μ L 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 hNGF β antibody. Spiral ganglion cells were stained with hematoxylin and eosin staining.
2.4
Detection of hNGF β expression
The hNGF β positive reaction product in the cochlea was detected by immunohistochemical EnVision method. The primary antibody we used is rabbit anti human hNGF β (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.
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-hNGF β target gene was transferred into left inner ears, hNGF β /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-hNGF β target gene with no blast exposure (hNGF β /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- μ L microinjector was inserted into the perilymph fluid and Ad-hNGF β (Institute of Clinical Medicine Yunyang School of Medicine, Yunyang, China) was injected slowly, 10 μ L per ear totaling 1 × 10 9 viruses for the hNGF β group, the hNGF β /blast group and the hNGF β /control group, and 10- μ L 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 hNGF β antibody. Spiral ganglion cells were stained with hematoxylin and eosin staining.
2.4
Detection of hNGF β expression
The hNGF β positive reaction product in the cochlea was detected by immunohistochemical EnVision method. The primary antibody we used is rabbit anti human hNGF β (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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