With the advance of industrial applications of radiation in modern society, humans are increasingly subjected to electromagnetic radiation. In modern warfare, electromagnetic radiation, especially high-power microwave (HPM), poses a growing threat to human health. However, whether and how electromagnetic radiation can affect cochlear hair cells remains unclear. HPM refers to electromagnetic waves with peak power exceeding 100 MW and frequencies ranging from 1 to 300 GHz, thus spanning centimeter and millimeter bands. The biological effects of HPM represent a new, hot research topic that has accompanied the development of HPM weapons. Indeed, the advent of HPM has greatly attracted attention to its biological effects, as resulting injuries are not only caused by direct exposure to microwave weaponry, but also by frequent low-dose HPM exposure and accidental high-dose HPM leaks occurring during microwave weapon production and training. Importantly, HPM contains some unique effects that are not readily observed with ordinary biological waves. To date, several studies have focused on the biological effects of microwaves on vital organs; however, no previous research examined the effects of HPM on the auditory system. Hence, it is essential to study the mechanisms by which HPM causes damage to cochlear hair cells. Abstract
HPM-induced damage to organisms has attracted significant attention form scholars. Indeed, previous studies have shown that HPM has harmful effects on several human organs and systems.[2,3] In particular, the nervous, cardiovascular, immune, and reproductive[6,7] systems are target organs that may be negatively affected by HPM, with adverse consequences such as nerve weakness, memory loss, myocardial injury, hematopoietic dysfunction, and declined sperm motility. The influence of microwave on auditory morphology has been gradually studied. Initially, Yu et al[8,9] found that a high-power electromagnetic pulse could lead to increased auditory brainstem response (ABR) thresholds in mice. Subsequently, Feng et al reported that prolonged exposure to microwaves could lead to dysfunction of outer hair cells. Later, Seckin et al demonstrated that radiofrequency radiation caused damage to cell structures of the cochlea during rat development. However, studies examining pathological changes elicited by HPM in cochlear hair cells and potential protective effects are still lacking. Hence, in this study, we established an HPM-induced hair cell injury model to observe the effects of HPM on guinea pig cochlear hair cells.
The zinc finger protein A20 was initially discovered in 1990 in human endothelial cells as a response product to tumor necrosis factor. Through gene sequencing, it was found that its readable frame encoded a new zinc finger protein, named zinc finger protein A20 or just A20 for brevity. It has been revealed that A20 can inhibit nuclear factor-kappa B (NF-κB) and caspase-3 expression to protect cells form damage,[13,14] such as apoptosis of endothelial cells and nerve cells. Auditory hair cells are the primary sensory receptors of the mammalian cochlea, irreversible damage to hair cells is an important cause of sensorineural deafness. Previous research has shown that hair cell damage is mainly caused by caspase-3 activation, release of reactive oxygen species, and NF-κB activation.[17,18] This is consistent with the anti-damage mechanism of A20, so we chose the zinc finger protein A20 for this initial examination of a protective effect in HPM-induced hair cell injury model. We constructed a recombinant adenovirus pAdEeay-1/A20 containing the zinc finger protein A20 gene to investigate potential protection of hair cells from HPM-induced damage. Adenovirus is an effective vector for gene therapy of inner ear, and does not cause damage to inner ear function. With regard to the importance of hearing both during peacetime and wartime, it is of great theoretical value and practical significance to study the effect of electromagnetic radiation on hearing function.
Materials and methods
In this study, 37 healthy guinea pigs of either sex (weighing 250 ± 25 g) were purchased from the Experimental Animal Center of the Third Military Medical University, Chongqing, China (license No. SCXK(Yu)20170002). Animals exhibited a normal auricle reflex and had no middle ear infection. This study was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University, China on April 18, 2017.
Animal grouping and establishment of HPM-induced hair cell injury model
Twelve guinea pigs were randomly assigned into groups (sham-exposed and HPM-irradiated groups) using a table of random digits. In the sham-exposed group (n = 3), also referred to as the control group, peak power density was 0W/cm2 for 20 minutes, and then experimental measures were observed. In the HPM-irradiated group (n = 3 guinea pigs in each group at each timepoint), peak power density was 65 W/cm2, which was applied for 20minutes, and findings were observed at 3, 6, and 12 hours after microwave irradiation.
Administration of recombinant adenovirus pAdEeay-1/A20 to cochlear hair cells
Twenty-five healthy guinea pigs were randomly assigned into five groups: normal control, pAdEeay-1, pAdEeay-1/irradiation, pAdEeay-1/A20/irradiation, and artificial perilymph/irradiation. Left cochlea from each guinea pig was chosen as the experimental ear and the other ear was given nothing as control. In the normal control group (n = 5), no treatment was performed. In the pAdEeay-1 group (n = 5), 5 μL of virus solution (Department of Anatomy, Third Military Medical University, Chongqing, China) was injected into left cochlea via 5 μL microsyringe. In pAdEeay-1/irradiation (n = 5) and pAdEeay-1/A20/irradiation (n = 5) groups (plasmid pCAGGS-FLAGmA20 was provided by the Anatomy Department of Third Military Medical University, Chongqing, China), 65 W/cm2 irradiation for 20 minutes was applied 3 days after injection of 5 μL of virus solution, and observations were made after 6 hours. In the artificial perilymph/irradiation group (n = 5), 65 W/cm2 irradiation for 20 minutes was administered 3 days after injection of 5 μL of artificial perilymph, and observations were made after 6 hours. Artificial perilymph was freshly prepared on the day before use in live animals, via pyrogen-free, sterile, double distilled water with the following salt and buffer concentrations: NaCl 125 mM, KCl 3.5 mM, MgCl2 1.20 mM, NaH2PO4 0.75 mM, NaHCO3 25 mM, glucose 5 mM, adjustment pH to 7.4.
Guinea pigs were placed into a microwave anechoic chamber with a reflection coefficient of approximately zero. The ambient temperature and humidity of the darkroom were kept constant by an air conditioner and dehumidifier, respectively, with a stable temperature of 20 ± 2°C and relative humidity of 60 ± 10%. Subsequently, guinea pigs were placed into a microwave-transparent box during irradiation, with consideration to both the long axis and direction of electric field polarization. microwave-transparent box is made of organic glass which can transmit electromagnetic radiation. Animals were irradiated with a microwave horn antenna at a peak power density of 65 W/cm2 in far-field (an irradiation system China Aim 5 Radar, provided by the Electromagnetic Radiation Laboratory of the Third Military Medical University, Chongqing, China), and the whole body was irradiated for 20 minutes at each experiment.
Recombinant virus transfection
In this study, adenovirus was selected as the transfer vector for the target gene, A20, which was cloned into the adenovirus expression vector and transfected into HEK 293 cells (American Type Culture Collection (ATCC), Manassas, VA, USA) for packaging. Virus titer was determined and an infectious recombinant virus pAdEeay-1/A20 with high titer was established, to achieve the goal of high expression of A20 gene in hair cells by gene transfection.
Guinea pigs were intraperitoneally anesthetized with 2% pentobarbital (30 mg/kg; Sigma). Cutting the skin and muscle behind the ear and opening of the bulla, the round window was exposed. Through the round window membrane, 5 μL of virus solution or artificial perilymph was slowly injected with a constant flow rate by 5 μL microsyringe. The coagulation piece was attached to the round window membrane, muscle tissue was adjusted to block the perforation, and the incision was sutured.
Testing of ABR threshold
All animals were tested for ABR threshold before and after irradiation, and administration of virus or artificial perilymph. Guinea pigs were administered general anesthesia (2% pentobarbital sodium, 30 mg/kg intraperitoneal injection) and the auditory acuity of animals was tested in an acoustic chamber using a brainstem auditory evoked potential detector (Nicolet Instrument, Danbury, CN, USA). The needle electrode was placed in the center of the forehead, the reference electrode in the contralateral mastoid process, and the grounding electrode in the upper lip, with an interelectrode resistance <2kΩ. An earphone plug was used. Stimuli included broadband pulsed sounds (0.1-ms pulse width, 100–3000 Hz bandwidth, 10-ms sweep duration, 256–512 superposition folds, and 100-dBSPL maximum peak level of stimuli). The stimulus intensity of wave III was recorded under various sound stimuli, and the sound stimulus was recorded as the acoustic threshold immediately after the disappearance of wave III.
Hair cell counting in stretched preparation of cochlea basilar membrane as detected by propidium iodide staining
Cochlea tissues were fixed with 4% paraformaldehyde, decalcified and dissected for the isolation of basilar membrane. Basilar membrane was stained with 100 μg/mL PI solution (Molecular Probes, Eugene, OR, USA) at room temperature in dark for 20 minutes. After washing samples four times for 5minutes in phosphate-buffered saline solution, tissues were spread onto glass slides and mounted with 50% glycerol. Under fluorescence microscopy (DH50; Nikon, Tokyo, Japan), hair cells from the basal turn of the cochlea basilar membrane that were undergoing apoptosis, swollen, or had absent nuclei were counted at 400× magnification within 0.2 mm of the tissue specimens.
Injected and contralateral non-injected cochleae (control) of guinea pigs in pAdEeay-1/A20/irradiation group were immunostained for A20 to detect transfection efficacy 3 days after pAdEeay-1/A20 injection. The cochleae were quickly harvested and perfused rapidly with 4% paraformaldehyde in phosphate-buffered saline and immersed in paraformaldehyde for 4 to 8 hours fixation at 4°C. Then, the specimens were immersed in 10% sucrose and 30% sucrose (each overnight) and then serial cryostat sections (6 μm thick) were cut with a Leica microtome (Leica CM1900, Germany) and placed on silane-coated slides. The tissue specimens with complete organ of Corti were incubated as follows: (1) normal goat serum (Solarbio, China) at room temperature for 30 minutes, (2) monoclonal rabbit anti-A20 antibody (1:200 diluted; #ab92324; Abcam, Cambridge, UK), overnight at 4°C, (3) FITC conjugated goat anti-rabbit antibodies (1:1000 diluted; #ab6717; Abcam), 30 minutes at room temperature. The specimens were examined with a fluorescence microscope (DH50; Olympus, Japan).
Scanning electron microscopy of cochlear hair cells
Effects of electromagnetic radiation on cochlear hair cells were analyzed by observing hair cell loss and changes in stereocilia by scanning electron microscopy (SEM). The cochleae were removed and gently perfused with 2.5% phosphate-buffered glutaraldehyde (catalog number G5882 MSDS; Sigma; pH 7.4) through the open round window and the cochlear apex. The cochleae remained in the same solution overnight. The bony capsule was removed after washing with 0.1 M phosphate-buffered saline. The spiral ligament and stria vascularis were removed under a dissecting microscope (XTL-3400; Shanghai Optical Instruments Import and Export Co., Ltd., Shanghai, China). The Reissner membrane was separated. The dissected specimens were rinsed with 0.1 M phosphate-buffered saline, post-fixed in 1% osmium tetroxide for 2 hours and placed in 2% tannic acid twice for 30 minutes. The cochleae were dehydrated in a series of graded ethanol solutions and dried in a critical point drier (hcp-2; Hitachi, Tokyo, Japan). The specimens were fixed on a metal stage, gold-coated in a sputter coater (E102 Ion Sputter; Hitachi) and observed under a SEM (S-4800; Hitachi).
Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). Data are expressed as the mean ± SD. Differences in ABR threshold and survival rates of inner and outer hair cells among the five groups were analyzed by one-way analysis of variance, two-samples t-test was used for the further paired comparison between two groups. A value of P < 0.05 was considered statistically significant.
Establishment of HPM-induced hair cell injury model
Threshold measurement results for evoked potentials of ABR
ABR threshold values for each group were measured at each time-point after irradiation (Table 1). There was no significant change in ABR threshold before and after irradiation in the control group (P > 0.05). Threshold changes in the HPM-irradiated group were not obvious after 3hours of irradiation. After 6 and 12hours of irradiation, the threshold was significantly higher than control group (P < 0.05, vs control group).
Numbers of survived hair cells in the cochlear basal turn of HPM-induced hair cell injury model animals
Light microscopy observations showed that there was no significant difference in the number of survived hair cells in the control group (Fig. 1A). In addition, there was no significant difference in the number of survived cochlear hair cells in the HPM-irradiated group after 3 hours of irradiation, although cells produced light-scattering and were partially swollen (Fig. 1B). After 6hours of irradiation, a significant loss of hair cells was observed (P < 0.01), and hair cells appeared swollen and fuzzy (Fig. 1C). Statistical changes of survived cochlear hair cells observed 6 hours after irradiation are presented in Table 2. The number of survived inner hair cells and outer hair cells was relatively lower in HPM irradiation group compared with control group.
Ultrastructure of hair cells
Results of SEM indicated that hair cells of control group were neatly arranged, the stereocilia were V-shaped and arranged in a row of upright clusters (Fig. 2A). After 6 hours of 65 W/cm2 irradiation, hair cells damage could be obviously observed. The stereocilia of hair cells were scattered or lost (Fig. 2B). After 12 hours of 65 W/cm2 irradiation, hair cell was severely damaged, and most of the stereocilia were scattered or lost (Fig. 2C). These results were consistent with light microscopy observations. Besides, in hair cells, especially inner hair cells, several irregular spherical objects of various sizes were observed, and their number was increased with the extended irradiation and the aggravation of damage (Fig. 2E and F).
Protective effects of zinc finger protein A20 on damaged hair cells
Threshold measurement results of ABR
Irradiation density of 65 W/cm2 was used as the study dose. The results (Table 3) showed that there was no significant change in hearing threshold after the administration of injections in any group (P > 0.05); moreover, there was no significant difference in ABR response threshold after injection of pAdEeay-1 in the pAdEeay-1 group (P > 0.05). These results revealed that administration of the recombinant adenovirus had no significant effect on animal ABR response threshold, nor did it cause inner ear injury. In the pAdEeay-1/irradiation group, ABR threshold was remarkably changed after irradiation (P < 0.01), suggesting that pAdEeay-1 had no protective effect on HPM-induced hearing loss. ABR thresholds of pAdEeay-1/A20/irradiation and artificial perilymph/irradiation groups were significantly increased compared with the normal control group (P < 0.05). However, the degree of ABR threshold increase in the pAdEeay-1/A20/irradiation group was lower than observed in the artificial perilymph/irradiation group (P < 0.01). These results indicated that A20 elicits a certain protective effect on HPM-induced hearing loss, although it is incomplete.
Number of cochlea hair cells
In this study, we first validation transfection efficacy of recombinant virus by detection A20 expression 3 days after pAdEeay-1/A20 injection in pAdEeay-1/A20/irradiation group. Immunofluorescence staining of frozen section of cochlea showed that there was no expression of green fluorescent protein in the contralateral non-injected cochleae (Fig. 3A), while green fluorescent protein was expressed in the inner hair cells and outer hair cells of the injected cochleae (Fig. 3B), indicating that recombinant adenovirus could transduce the target gene A20 in hair cells. Then four types of hair cells (normal, apoptotic, necrotic, and missing) were observed after PI staining of fixed cochlear basal membranes. Apoptotic, swollen, and lost cells were counted as the number of damaged hair cells. Statistical analysis (Table 4) showed that there was no significant difference in numbers of survived hair cells between control group and pAdEeay-1 group, which was consistent with light microscopy observations (Fig. 3C and D), indicating that intracochlear drug injections through the round window membrane did not cause morphological changes. However, there were significant differences in numbers of survived hair cells in pAdEeay-1/irradiation, pAdEeay-1/A20/irradiation, artificial perilymph/irradiation, and control groups (P < 0.05), indicating damage was existed in hair cells in these 3 groups. Nevertheless, the number of survived hair cells in the pAdEeay-1/A20/irradiation group was larger compared with pAdEeay-1/irradiation and artificial perilymph/irradiation groups (Fig. 3E–G), suggesting that A20 had a protective effect on HPM-induced hair cell damage, although this protection was incomplete.
Ultrastructure of hair cells damaged by HPM
In the control group, hair cells of the cochlear basilar membrane were neatly arranged, while stereocilia were arranged in upright clusters without scattered or missing components (Fig. 4A). No significant damage was found in pAdEeay-1 group (Fig. 4B). Partial cilia of outer hair cells were dislodged or missing in the organ of Corti of the artificial perilymph/irradiation group. Some inner hair cells exhibited spherical objects of varying sizes and irregular shapes (Fig. 4C). In the pAdEeay-1/A20/irradiation group, observed injuries were significantly reduced compared with the artificial perilymph/irradiation group, part of the hair cells showed stereocilia collapsed and fused stereocilia. However, no significant decrease in spherical objects was observed (Fig. 4D). Indicating that A20 elicits a certain protective effect on HPM-induced hair cells damage, although it is incomplete.
Damage induced by HPM has attracted significant attention from scholars. The nervous, cardiovascular, immune, and reproductive systems are all target organs of HPM. The detrimental effects of electromagnetic radiation are mainly manifested in two aspects: thermal and non-thermal biological effects. In this study, we found that HPM radiation with different power densities could induce autonomous changes in guinea pigs during irradiation. With increased power density and duration of radiation, the process of excitation followed by inhibition was observed. Rectal temperature of guinea pigs was significantly increased after irradiation, indicating that the thermal effect was likely related to changes in the guinea pig nervous system (data not shown). Therefore, it was revealed that guinea pigs were indeed affected by HPM irradiation, similar to other animals.[21,22] However, whether electromagnetic radiation, especially HPM, has local effects on the inner ear had not been reported. Our previous study showed that with a radiation density of 30 W/cm2, the rectal temperature of guinea pigs increased after 20 minutes of irradiation, and they showed increased scratching, limb licking, and disturbance activities. However, no significant changes were observed with regard to hair cell damage at 3, 6, and 12 hours according to propidium iodide staining and SEM results. Moreover, there was no significant change in ABR threshold values after irradiation, indicating that changes in the nervous system of guinea pigs during irradiation were observed earlier than changes in the auditory system. With a radiation density of 65 W/cm2, the overall effect on guinea pigs were particularly significant after 20minutes of irradiation, as animals exhibited shortness of breath, hyperactivity, sweating, and listlessness. Subsequent PI staining showed that the morphology of hair cells was not significantly changed after 3hours, although there was obvious damage after 6hours including a significantly increased number of damaged hair cells compared with 6hours in the control group. Additionally, loss of inner hair cell stereocilia, spherical substances around inner hair cells, and damaged outer hair cell stereocilia were observed by SEM. The number of damaged hair cells was increased after 12hours compared with 6hours, and ABR detection results reflected similar changes. With a radiation density of 90 W/cm2, the above-mentioned changes were more obvious. In our preliminary experiment, some guinea pigs died after 90 W/cm2 of irradiation, indicating that guinea pigs had poor tolerance to this rate, which was not appropriate for further experimental studies. Collectively, these results indicated that HPM can damage hair cells, and the effect of damage increases with irradiation density. It is noteworthy that HPM primarily damages inner hair cells, which exhibited irregular spherical substances around them. The composition and efficacy of these spheres remains unclear, highlighting the necessity of further studies. Moreover, with an irradiation density of 30 W/cm2, there was a thermal effect in guinea pigs; thus, while there was no significant change in hair cell morphology or ABR. So, irradiation density of 65W/cm2 was used as the study dose.
In this study, it was revealed that HPM can cause damage to inner ear hair cells, which is important as irreversible damage to inner ear hair cells is an important cause of sensorineural deafness. At present, there is no effective therapeutic method to repair or replace damaged hair cells. Thus, preventing hair cells from incurring further damage is of great importance for the prevention of hearing loss. It has been revealed that A20 can inhibit NF-κB and caspase-3 expression to protect cells form damage. Therefore, zinc finger protein A20 is a good candidate for hair cell protection. Herein, we presented the recombinant adenovirus pAdEeay-1/A20, which was successfully injected via the round window into guinea pigs cochlea, whereby the recombinant adenovirus A20 gene was effectively expressed by hair cells.
It has been found that hair cells in the basal turn are more sensitive to cytotoxic effects induced by noise, age, and other factors compared with hair cells in the median or apical turns.[23–25] Therefore, we selected hair cells in the basal turn to examine damage and protective effects. Now, we find that there was no significant difference in ABR threshold between control and pAdEeay-1 groups after cochlear administration, indicating that administration of recombinant adenovirus did not injure the inner ear. However, there were significant changes in hearing of the pAdEeay-1/irradiation group, suggesting that pAdEeay-1 had no protective effect on hearing loss. The ABR threshold of pAdEeay-1/A20/irradiation and artificial perilymph/irradiation groups was significantly reduced compared with the normal control group. In addition, the ABR threshold of the PAdEeay-1/A20/irradiation group was significantly lower than the PAdEeay-1/irradiation group and artificial perilymph/HPM group, suggesting that A20 had a protective effect on hearing loss caused by HPM ototoxicity, although this effect was incomplete. Morphologically, pAdEeay-1/A20 may also play a protective role in hair cells which can reduce HPM-induced damage primarily manifested as apoptosis, necrosis, and loss of inner hair cells. Overall, A20 could protect hair cells from HPM-induced damage to prevent further hearing loss.
However, there were some unexplained phenomena in our research. For example, what were the components of globules observed in the HPM injury group? In addition, as pAdEeay-1/A20 is typically expressed in a variety of cells in the cochlea, it can be concluded that there are interactions between those cells. Finally, whether pAdEeay-1/A20 directly or indirectly protects hair cells, or elicits further effects on other cells remains unclear. Therefore, it is essential to further clarify these unknown questions in future experiments.
WY conceived and designed the experiments. FT, XC, and HL performed the experiments. JL and LJ analyzed the data. FT and XC wrote the manuscript. All authors approved the final manuscript.
This work was supported by grants from the National Natural Science Foundation of China (No. 81873702, 81470694; to WY), Chongqing Natural Science Foundation of China (No. cstc2017jcyjAX0407; to WY), Southwest Hospital Foundation of China (No. SWH2016JCZD-02; to WY).
Institutional review board statement
The study was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University on April 18, 2017.
Conflicts of interest
The authors declare that they have no conflicts of interest.
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Keywords:Copyright © 2019 The Chinese Medical Association. Published by Wolters Kluwer Health, Inc.
cochlea; zinc finger protein A20; high power microwave; hair cells