Animals and Study Groups
One-month-old C57BL/6 female mice weighing 25-27 grams were purchased from the Experimental Animal Center of Shandong University for this experiment, and all animal procedures in this study were approved by the Ethics Committee of the Second Hospital of Shandong University (Permit Number: KYLL-2016(GJ)A-0001).
Study Groups: 48 mice were randomly divided into four groups: a wild type group (Sham/WT), RvE1-pretreated wild type group (RvE1), X-ray irradiation group (Rad), and RvE1-pretreated X-ray irradiation group (Rad+RvE1). On days 7 and 14 after irradiation, six mice in each group were sacrificed for experimental analysis.
Irradiation method: experimental mice were irradiated with an Elekta Synergy medical linear accelerator (Elekta, Stockholm, Sweden), 6 MV-X ray, irradiating the inner ear from both sides with the source axis distance (SAD = 100 cm) technique. The irradiation depth of the ear was determined by thin computed tomography (CT) scan with a sample mouse. The irradiation field was 1 cm × 1 cm in size, which fully enveloped the inner ear. The thickness of the lead block was 10 cm, and the radiometric rate was 400 cGy/min.
The irradiation groups were irradiated with 20 Gy in one fraction to the inner ear. This dose was determined according to the results of a preliminary experiment, to ensure robust survival of all experimental mice along with detectable damage to inner ear functions. Before irradiation, 10% chloral hydrate (the Second Hospital of Shandong University, Jinan, China) was used for enterocoelia anesthesia at a dose of 1,000 mg/kg without any signs of peritonitis after administration. On the day of the experiments, 1 μg RvE1 (Cayman Chemical, Ann Arbor, USA) was dissolved in 1mL isotonic saline and administered to the mice via intraperitoneal injection 30 minutes before irradiation, according to a previous report [11,16].
Auditory Brainstem Response (ABR)
After the experimental mice were anesthetized as described above, they were placed in a soundproof room, the collection electrode was inserted into the top of the skull, the reference electrode was inserted into the mastoid process, and the ground electrode was inserted into the back midline of the mouse. The external speaker was moved to the ear lobe, and clicks and pure tones were performed successively. The selected pure tone frequencies were 4, 8, 16, and 32 kHz, and the results were recorded with a High-frequency Auditory Signal Processor RZ6 Multi-I/O Processor (Tucker Davis Technologies, Alachua, FL, USA) for statistical analysis. ABR tests were performed before irradiation and also on days 7 and 14 after irradiation.
Roller Test
In this test, each mouse was first acclimated to the roller activity for 3 days, ten minutes per day, then on the day of the experiments, the same ten-minute roller activity was performed 30 minutes before the balance time was officially recorded. The roller speed was 10 r/min during training and testing. During the test, the mice were placed on a rotating roller in turn, and the duration that each mouse stayed on the roller was recorded for three replicates and averaged. Relative balance ability was calculated from the balance time of each mouse on day 7 or day 14 divided by the balance time on the first day before treatment. This test was performed with a Mouse Swivel Fatigue Tester (Biowill Co., Huaibei, China)
Hematoxylin-eosin (HE) Staining
Mice were sacrificed by cervical dislocation, and the cochlea was removed as soon as possible, transferred to precooled 4% PFA and perfused. The cochlea tissue was placed at 4°C overnight and then washed three times with 1× PBS for 10 minutes each, followed by 10% EDTA for decalcification. One week later, cochleae were washed with 1× PBS and processed for paraffin embedding. The cochleae were then embedded and sectioned at 4-μm thickness and stained with H&E.
Whole-mount Staining
Cochleae were decalcified in 10% EDTA for 3 days and washed 3 times with 1× PBS for 10 minutes each. The basal membrane and vestibule were obtained by dissecting the cochleae and then permeabilized with 0.2% Trition X-100 for 8 minutes. The basal membrane and vestibule were then blocked for 30 minutes with 10% normal goat serum (NGS). The Myosin VIIa (myo7a) antibody (Abcam, Cambridge, MA, USA), diluted 1:200, was added to the basal membrane and vestibule and left overnight at 4°C. The membrane and vestibule were then washed and incubated with 1:200 mouse-IgG 594 (Bethyl, Montgomery, AL, USA) for 1 hour and then with 1:500 phalloidin (Sigma Aldrich, St Louis, MO, USA) for 40 minutes. After washing with 1× PBS 3 times, DAPI (Sigma Aldrich) at a 1:250 dilution was added and incubated for 10 minutes at room temperature. The specimens were sealed by glycerin, and organs of Corti, spiral ganglion, utricle and sacculus were imaged using a confocal microscope (LSM700; Carl Zeiss, Germany).
Scanning Electron Microscopy (SEM)
Cochleae were decalcified in 10% EDTA for 3 days and then soaked in precooled 2.5% glutaraldehyde (Beijing Dingguo Changsheng Biotechnology Co. LTD, Beijing, China). Cochleae were dissected to obtain the basal membrane and vestibule, which were washed three times with 1× PBS for 10 minutes each. Then, osmium tetroxide was added in a fume hood. Samples were washed again three times with 1× PBS and 4 times with sterile distilled water, before dehydrating through an ethanol gradient. Then, the critical point was selected for drying the sample with carbon dioxide for 2 hours. Dried specimens were sputter coated with gold for 4 minutes. Following the preparation process, the samples were observed under a scanning electron microscope (Zeiss sigma 300, Carl Zeiss, Germany).
Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
At the indicated days, mice were sacrificed for dissection of the basal membrane, modiolus, and vestibule. RNA was extracted from each tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using a mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. To ensure that total RNAs met the requirements for the experiment, the purity and concentration of RNA were quantified using the Nano Drop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the integrity of RNA was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total cDNA was synthesized using a one-step cDNA synthesis kit (Vazyme, Nanjing, China). Using cDNA as a template, changes in the expression of inflammatory factors (IL-2, IL-6, IL-10, tumor necrosis factor [TNF]-α, and interferon [IFN]-γ) in the tissues were analyzed by qRT-PCR. The qRT-PCR reactions were carried out using a 7900 HT Fast RealTime PCR system (Applied Biosystems, Foster City, CA, USA) and the reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 68°C for 25 seconds, 40 cycles; 72°C extension for 5 minutes, and hold at 4°C. Relative gene expression was calculated using the comparative 2-△△Ct method with the housekeeping gene mus-β-actin as a reference. All primers were synthesized by Invitrogen and sequences were as follows: IL-2 forward primer: 5’-AGATGAACTTGGACCTCTGCG-3’, reverse primer: 5’-AAAGTCCACCACAGTTGCTG-3’ (175bp). IL-6 forward primer: 5’-TTCCTCTGGTCTTCTGGAGT-3’, reverse primer: 5’-GAGAGCATTGGAAATTGGGGT-3’ (181bp). IL-10 forward primer: 5’-AGCCTTATCGGAAATGATCCAGT-3’, reverse primer: 5’-GGCCTTGTAGACACCTTGGT-3’ (229bp). TNF-α forward primer: 5’-CCTGTAGCCCACGTCGTAG-3’, reverse primer: 5’-GGGAGTAGACAAGGTACAACCC-3’ (148bp). IFN-γ forward primer: 5’-ACAGCAAGGCGAAAAAGGATG-3’, reverse primer: 5’-TGGTGGACCACTCGGATGA-3’ (106bp). β-actin forward primer: 5’-ACGGCCAGGTCATCACTATTG-3’, reverse primer: 5’-AGGGGCCGGACTCATCGTA-3’ (372bp).
Statistical analysis
Statistical analysis was performed using SPSS 20.0 software (IBM SPSS Statistics for Windows, Armonk, NY, USA). The expression levels of inflammatory factors, hearing threshold and relative balance ability were expressed as the mean ± standard deviation (SD) and the differences among different groups were compared by one-way analysis of variance (ANOVA), wihle multiple comparisons among different subgroups were tested using the Bonferroni test. All P-values were two-sided, and a value of P <0.05 was considered statistically significant.