Abstract
Objective
Magnesium is proved to attenuate acoustic trauma, and reactive oxygen species (ROS) formation is a critical role that involves hearing loss induced by impulse noise. We aimed to investigate the relationship between the cochlea magnesium content, ROS formation and hearing loss induced by impulse noise.
Methods
Ninety pigmented guinea pigs were exposed to impulse noise. Auditory thresholds were assessed by sound-evoked auditory brainstem response (ABR) 24 h before and 72 h after exposure to impulse noise. 4-Hydroxynonenal(HNE) used as a marker of ROS was determined immunohistochemically. The cochlea magnesium content was examined with the method of energy dispersive x-ray analysis, and the cochlea was also detected with scanning electron microscope. The relationship between the cochlea magnesium content, ROS formation and hearing loss was analyzed.
Results
There was loss of outer hair cell cilia accompanying with significant auditory threshold shift after impulse noise exposure. ROS was positive in the organ of Corti of all animals. The cochlea magnesium content was negatively correlated with ROS formation and hearing loss.
Conclusions
Inhibiting ROS formation is one of the mechanisms for magnesium to reduce acoustic trauma, and difference in cochlea magnesium contents is one of the factors that induce varying degrees of cochlear damage among each individual after acoustic trauma.
1
Introduction
Impulse noise from firearms in recreational and military environments forms one of the greatest hazards of acute intense acoustic trauma. Excessive impulse noise exposure inflicts both mechanical and metabolic damage to the inner ear, which has a significant impact on the quality of life and may lead to noise-induced hearing loss (NIHL). In humans, over damage of cochlea is permanent, as the auditory cells are without the ability of regeneration. Military noise-induced NIHL continues to be a severe and a costly problem despite hearing conservation programs. Even before World War I, military veterans were receiving compensation for NIHL. The medical records of Union Army soldiers demonstrate that 33% of military men had been diagnosed as NIHL . Soldiers with NIHL from their military service were guaranteed a larger pension as compensation.
Mechanical damage of cells in the organ of Corti was confirmed to be the primary cause of impulse noise-induced hearing loss. After mechanical damage, metabolic disorders occurred. Metabolic disorders have multiple origins: ionic, ischemic, excitotoxic and production of cochlear free radicals causing cell death, due to necrosis or apoptosis. Many researches focused on free radicals and lipid peroxidation products, and the use of antioxidant agents holds significant therapeutic promise for NIHL . Recently, much attention has been paid for magnesium therapy on NIHL. One-month treatment with magnesium after an impulse noise trauma was effective in the preservation of hair cell and cochlear function. It suggests that magnesium acts on the later metabolic processes after noise exposure . Magnesium, a small molecule that can easily cross the blood labyrinth barrier, presents fully its neuroprotective and vasodilatory effects in cochlea. The efficacy of magnesium, administered either to prevent or to treat NIHL has been demonstrated in several studies in animals and humans . It is general consensus that magnesium can attenuate NIHL.
It is a recognized knowledge that cochlear damage in different people who are exposed to the same impulse noise has varying degrees. Although in the same live fire training, audiological investigation showed that some soldiers were with no cochlear trauma, and some with severe cochlear damage. Also, some soldiers recovered rapidly from impulse noise damage, and some appeared to have permanent hearing loss. Different responses to noise depend mainly on susceptibility gene , just like genetic susceptibility to aminoglycoside ototoxicity. As magnesium can attenuate NIHL, we guess that the cochlea magnesium content may also contribute to varying responses to impulse noise. The aim of the present study was therefore to explore the relationship between the cochlea magnesium content and impulse noise trauma.
2
Materials and methods
2.1
Animals and impulsive noise exposure
Ninety pigmented guinea pigs weighing between 250 and 350 g were used in this study. All experimental protocols were reviewed by the Committee for Ethics on Animal Experiments of Guangzhou General Hospital of Guangzhou Military Command (Guangzhou, China). All experiments were carried out in accordance with these guidelines. All invasive procedures were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. All animals were confirmed to have a positive Preyer’s reflex and were microscopically examined to be free from otitis media. They were free to move around inside the cages and had free access to normal food and water at all times. The animals were exposed to intense impulse noise from the 7.62-mm Chinese Army 81-1 type of assault rifle. Each animal was exposed to impulses of 15 shots delivered at an interval of 1 s to avoid the involvement of the stapedius reflex. During the exposure each animal’s head was kept toward the muzzle of the rifle at the distance of 0.35 m to the direction left 15° (impulse peak 176 dB SPL, frequency spectrum 1.05–20.3 kHz). Sound peak was measured by digital sound pressure meter (TDJ-23, Jinan, China). Impulse peak had an average of 10 shots.
2.2
Auditory brainstem response measurements
The guinea pigs were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). ABR recordings were obtained 24 h before and 72 h after exposure to impulse noise by means of an electrodiagnostic system (Pathfinder I; Nicolet Biomedical Instruments). The responses were recorded in the far-field technique. The reference electrode was inserted s.c. into the subcutaneous below the ipsilateral pinna, the ground electrode into the subcutaneous below contralateral pinna and the active electrode into the top of the head, respectively. Acoustic stimuli were delivered by an earphone through a small tube inserted into the external ear meatus in a sound-proof box. The stimuli consisted of click and tone bursts of 8, 16 and 32 kHz (sine wave pulses with a trapezoidal envelope, the total duration was 10 ms, and the rise and fall times were 2 ms). They were presented at a rate of 11.1 s − 1 and a duration of 0.11 ms. Responses were accumulated 500 times. The levels of stimuli were lowered from 95 to 10 dB SPL by 5 dB steps. The ABR threshold was determined as the minimum sound level giving reproducible waveforms. The recordings were repeated twice at the threshold level and reproducibility was confirmed.
2.3
Immunohistochemical investigation
All animals were sacrificed 72 h after exposure to impulse noise. In each animal, left cochlea was for immunohistochemical investigation, and right cochlea was for scanning electron microscopy. The tissues were fixed via cardiac perfusion with 4% (wt/vol) paraformaldehyde (pH 7.4) after flushing out the blood with 0.1 M PBS. The cochleae for immunohistochemical investigation were incubated in the same fixative overnight. Decalcification was performed with 10% (wt/vol) EDTA solution in Tris at pH 7.4 for 10 days. Subsequently, the tissues were embedded in paraffin. Each paraffin-embedded specimen was sectioned at a thickness of 6 μm with a microtome. The paraffin was removed by immersion in graded series of ethanol. Then the sections were immersed in 3% (vol/vol) H 2 O 2 for 20 min, followed by 0.25% (vol/vol) Triton X for 10 min. Subsequently they were incubated at 4 °C overnight with the primary antibody/anti-4-hydroxynonenal (4HNE) at 1:300 dilution (rabbit polyclonal antibody; LSBio). In some sections, incubation with the primary antibody was omitted. These sections served as a proof to specificity of the secondary antibodies. After rinsing with 0.1% (wt/vol) Tris–PBS solution (pH 7.4) and treatment with 3% (vol/vol) normal goat serum, the sections were incubated with the second antibody at 1:500 dilution (anti-rabbit; Sigma) for accentuation. The reaction was developed with a horseradish peroxidase (HRP) complex at 1:100 dilution for 1 h (Sigma) and a nickel-enhanced DAB (Sigma). The samples were dehydrated and mounted. Subsequent analysis and photography of the immunohistochemical expression pattern and intensity were conducted under a light microscope. Omitting the first antibody acted as the negative control, and the sections of cochlea damaged by cisplatin were used for positive control. The intensity of immunostaining in Corti’s organ was evaluated by density detected by Image-pro Plus software. The image of Corti’s organ was changed in the computer under microscopic vision. The magnification of every section was × 100. The brightness of the microscope was fixed at moderate for examination of every section. The Corti’s organ in every section was detected in the same condition. The density of immunostaining was automatically generated after drawing the boundary of the Corti’s organ. The immunostaining density detected by Image-pro Plus software is a relative value. All density evaluations of the Corti’s organs were detected in the same condition, so the results were comparable. Twenty sections were chosen randomly in every cochlea; the density of immunostaining in the Corti’s organs of every section was detected in the basal turn, second turn, and apical turn, respectively. The density of immunostaining in the Corti’s organ of each cochlea had an average of 20 sections. All analyses of immunostaining were carried out in a randomized double-blind manner.
2.4
Scanning electron microscope
After the cochleae were dissected, specimens were perfused immediately with 2.5% glutaraldehyde at 4 °C in 0.1 mol/L cacodylate (Cac) buffer (pH 7.4). A straight pick was used to create small openings into the round window, oval window, and apex of the cochlea through which the perfusions were performed. The specimens were then immersed in the glutaraldehyde solution and refrigerated overnight. The following day, the specimens were carefully rinsed with Cac buffer, and then postfixed with 1.5% osmium tetroxide in 1.0 mol/L Cac buffer. After 15 min of rotation the specimens were again infused with Cac buffer. The lateral wall was exposed under magnification by removing the bony capsule and lateral wall of the cochlea with a dental drill. The Corti organ was then exposed using a razor to dissect the spiral ligament. The specimens were dried, mounted, and sputter coated with gold palladium alloy. Photographs were taken using a Hitachi S-3000 N scanning electron microscope. Under the vision of scanning electron microscope, 10 different areas were chosen randomly in basal turn, second turn and apical turn respectively in every cochlea. Magnification of every area was × 1200. The number of outer hair cells with stereocilia loss was accounted in every area, and the rate of outer hair cells with stereocilia loss was calculated. The rate of outer hair cells with stereocilia loss in cochlear turn has an average of 10 different areas.
2.5
Energy dispersive x-ray analysis
For magnesium analysis, Hitachi S-3000N scanning electron microscope was equipped with an energy dispersive x-ray attachment (EMAX, Horiba, Japan). Magnesium content in 10 different areas which were chosen randomly in basal turn, second turn and apical turn respectively in every cochlea was detected by EMAX. The magnesium content in cochlear turn had an average of 10 different areas, and the magnesium content in cochlea had an average of basal turn, second turn and apical turn. In addition, there were no significant differences in magnesium content among cochlear turns. The magnesium content was expressed as weight percentage.
2.6
Statistical analyses
Correlation analyses were performed using a commercial statistical software package (SPSS 19.0).
2
Materials and methods
2.1
Animals and impulsive noise exposure
Ninety pigmented guinea pigs weighing between 250 and 350 g were used in this study. All experimental protocols were reviewed by the Committee for Ethics on Animal Experiments of Guangzhou General Hospital of Guangzhou Military Command (Guangzhou, China). All experiments were carried out in accordance with these guidelines. All invasive procedures were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. All animals were confirmed to have a positive Preyer’s reflex and were microscopically examined to be free from otitis media. They were free to move around inside the cages and had free access to normal food and water at all times. The animals were exposed to intense impulse noise from the 7.62-mm Chinese Army 81-1 type of assault rifle. Each animal was exposed to impulses of 15 shots delivered at an interval of 1 s to avoid the involvement of the stapedius reflex. During the exposure each animal’s head was kept toward the muzzle of the rifle at the distance of 0.35 m to the direction left 15° (impulse peak 176 dB SPL, frequency spectrum 1.05–20.3 kHz). Sound peak was measured by digital sound pressure meter (TDJ-23, Jinan, China). Impulse peak had an average of 10 shots.
2.2
Auditory brainstem response measurements
The guinea pigs were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). ABR recordings were obtained 24 h before and 72 h after exposure to impulse noise by means of an electrodiagnostic system (Pathfinder I; Nicolet Biomedical Instruments). The responses were recorded in the far-field technique. The reference electrode was inserted s.c. into the subcutaneous below the ipsilateral pinna, the ground electrode into the subcutaneous below contralateral pinna and the active electrode into the top of the head, respectively. Acoustic stimuli were delivered by an earphone through a small tube inserted into the external ear meatus in a sound-proof box. The stimuli consisted of click and tone bursts of 8, 16 and 32 kHz (sine wave pulses with a trapezoidal envelope, the total duration was 10 ms, and the rise and fall times were 2 ms). They were presented at a rate of 11.1 s − 1 and a duration of 0.11 ms. Responses were accumulated 500 times. The levels of stimuli were lowered from 95 to 10 dB SPL by 5 dB steps. The ABR threshold was determined as the minimum sound level giving reproducible waveforms. The recordings were repeated twice at the threshold level and reproducibility was confirmed.
2.3
Immunohistochemical investigation
All animals were sacrificed 72 h after exposure to impulse noise. In each animal, left cochlea was for immunohistochemical investigation, and right cochlea was for scanning electron microscopy. The tissues were fixed via cardiac perfusion with 4% (wt/vol) paraformaldehyde (pH 7.4) after flushing out the blood with 0.1 M PBS. The cochleae for immunohistochemical investigation were incubated in the same fixative overnight. Decalcification was performed with 10% (wt/vol) EDTA solution in Tris at pH 7.4 for 10 days. Subsequently, the tissues were embedded in paraffin. Each paraffin-embedded specimen was sectioned at a thickness of 6 μm with a microtome. The paraffin was removed by immersion in graded series of ethanol. Then the sections were immersed in 3% (vol/vol) H 2 O 2 for 20 min, followed by 0.25% (vol/vol) Triton X for 10 min. Subsequently they were incubated at 4 °C overnight with the primary antibody/anti-4-hydroxynonenal (4HNE) at 1:300 dilution (rabbit polyclonal antibody; LSBio). In some sections, incubation with the primary antibody was omitted. These sections served as a proof to specificity of the secondary antibodies. After rinsing with 0.1% (wt/vol) Tris–PBS solution (pH 7.4) and treatment with 3% (vol/vol) normal goat serum, the sections were incubated with the second antibody at 1:500 dilution (anti-rabbit; Sigma) for accentuation. The reaction was developed with a horseradish peroxidase (HRP) complex at 1:100 dilution for 1 h (Sigma) and a nickel-enhanced DAB (Sigma). The samples were dehydrated and mounted. Subsequent analysis and photography of the immunohistochemical expression pattern and intensity were conducted under a light microscope. Omitting the first antibody acted as the negative control, and the sections of cochlea damaged by cisplatin were used for positive control. The intensity of immunostaining in Corti’s organ was evaluated by density detected by Image-pro Plus software. The image of Corti’s organ was changed in the computer under microscopic vision. The magnification of every section was × 100. The brightness of the microscope was fixed at moderate for examination of every section. The Corti’s organ in every section was detected in the same condition. The density of immunostaining was automatically generated after drawing the boundary of the Corti’s organ. The immunostaining density detected by Image-pro Plus software is a relative value. All density evaluations of the Corti’s organs were detected in the same condition, so the results were comparable. Twenty sections were chosen randomly in every cochlea; the density of immunostaining in the Corti’s organs of every section was detected in the basal turn, second turn, and apical turn, respectively. The density of immunostaining in the Corti’s organ of each cochlea had an average of 20 sections. All analyses of immunostaining were carried out in a randomized double-blind manner.
2.4
Scanning electron microscope
After the cochleae were dissected, specimens were perfused immediately with 2.5% glutaraldehyde at 4 °C in 0.1 mol/L cacodylate (Cac) buffer (pH 7.4). A straight pick was used to create small openings into the round window, oval window, and apex of the cochlea through which the perfusions were performed. The specimens were then immersed in the glutaraldehyde solution and refrigerated overnight. The following day, the specimens were carefully rinsed with Cac buffer, and then postfixed with 1.5% osmium tetroxide in 1.0 mol/L Cac buffer. After 15 min of rotation the specimens were again infused with Cac buffer. The lateral wall was exposed under magnification by removing the bony capsule and lateral wall of the cochlea with a dental drill. The Corti organ was then exposed using a razor to dissect the spiral ligament. The specimens were dried, mounted, and sputter coated with gold palladium alloy. Photographs were taken using a Hitachi S-3000 N scanning electron microscope. Under the vision of scanning electron microscope, 10 different areas were chosen randomly in basal turn, second turn and apical turn respectively in every cochlea. Magnification of every area was × 1200. The number of outer hair cells with stereocilia loss was accounted in every area, and the rate of outer hair cells with stereocilia loss was calculated. The rate of outer hair cells with stereocilia loss in cochlear turn has an average of 10 different areas.
2.5
Energy dispersive x-ray analysis
For magnesium analysis, Hitachi S-3000N scanning electron microscope was equipped with an energy dispersive x-ray attachment (EMAX, Horiba, Japan). Magnesium content in 10 different areas which were chosen randomly in basal turn, second turn and apical turn respectively in every cochlea was detected by EMAX. The magnesium content in cochlear turn had an average of 10 different areas, and the magnesium content in cochlea had an average of basal turn, second turn and apical turn. In addition, there were no significant differences in magnesium content among cochlear turns. The magnesium content was expressed as weight percentage.
2.6
Statistical analyses
Correlation analyses were performed using a commercial statistical software package (SPSS 19.0).
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