Animals

Adult Sprague Dawley (SD) rats weighing 180–220 g were purchased from Vital River (Beijing, China). Animals were randomly divided into experimental and control groups. The rats were maintained in ventilated cages on a 12–12 light/dark cycle, temperature (23 °C ± 2 °C) standard laboratory conditions and given ad libitum access to food and water throughout the study at Capital Medical University. Rats were used for behavioral tests 0 day/7 day/14 day/1 month/3 month postoperatively. The animal data reporting followed the ARRIVE 2.0 guideline [15].

JVL rat model

JVL model was generated as previously reported [16, 17]. Briefly, rats were deeply anesthetized with 4% isoflurane and subsequently maintained between 1.5 and 2% isoflurane during the surgical procedure using an isoflurane vaporizer (RWD, Shenzhen, China). Each rat was placed in a dorsally recumbent position under a dissection microscope, and the hair from the neck was removed. A cutaneous midline incision was made on the neck. Blunt dissection of the subcutaneous tissue was performed, and the dorsolateral salivary glands were separated. The internal jugular vein, located in the carotid sheath on both sides (above the internal carotid arteries next to the vagus nerve), was subsequently separated and exposed according to the published protocol of Auletta and co-workers [17], making an oblique incision cross one-third point on the orbital line near the ear, retracting the temporalis muscle dorsally and the exorbital lacrimal gland ventrally, exposing and ligating the retroglenoid vein, which is the extracranial termination of the transverse sinus in rodents. Surgical ligations were performed with 8 − 0 nonabsorbable surgical sutures. Sham rats received the same skin incision and operation without surgical ligation under isoflurane anesthesia. Once the surgery was completed, the wounds were closed using a 6 − 0 surgical suture with a simple continuous pattern. Rats were allowed to recover under the heat lamp, and antibiotic ointment was applied over the skin incision, following five consecutive days. All efforts were made to minimize the number of animals used and their suffering.

Auditory brainstem responses (ABR)

Rats were deeply anesthetized with ketamine (100 mg/kg, i.p.) plus xylazine (10 mg/kg, i.p.). Needle electrodes were positioned subcutaneously beneath the pinna of the test ear (reference) and contralateral ear (ground) as well as at the vertex (active). Acoustic stimuli were elicited and evaluated by the Tucker Davis Technologies (TDT) System III hardware and SigGenRZ software (TDT, Alachua, FL, USA). Tone-burst stimuli were presented in 10 dB decrements from 90 dB sound pressure level (SPL) and the responses evoked were recorded at octave frequencies of 4, 8, 16, and 32 kHz. The ABR threshold was defined as the lowest stimulus intensity capable of producing repeatable (in at least two trials) ABR waves.

Gap prepulse inhibition of acoustic startle (GPIAS)

The GPIAS was performed using the Acoustic Startle Reflex Starter Package for Rat (Med Associates, St. Albans, VT, USA), as described previously [18, 19]. In brief, GPIAS sessions consist of 30 gap and 30 non-gap trials. Conscious rats experienced testing with different band-pass-filtered sounds (1k Hz bandwidth centered at 4, 8, 12, and 16 kHz, respectively) at 65 dB SPL. Startle responses were elicited by a burst of white noise (20 ms, in an interval of 30–35 s randomly) at 110 dB SPL. The gap in the narrowband noise began 100 ms before the acoustic startle stimulus (50 ms, 5 ms rise/fall time). The interval between each startling noise was 30–35 s and each test took approximately 30 min.

18 F-fluorodeoxyglucose (18 F-FDG) microPET

The Department of Medical Imaging Research at the Central Laboratory of Capital Medical University provided the 18 F-FDG used in this study. Before the scan, all the rats were fasted (but with free access to water) for 12 h to maximize FDG uptake in the brain; the body weight of each rat and radioactivity of the syringe (before and after injection) were recorded for calibration of the effective dosage. After an uptake period of approximately 45 min following the intravenous 18 F-FDG injection through the caudal vein, the PET signals and hybrid computed tomography (CT) data were obtained with an Inveon MM micro-PET/CT scanner (Siemens Co., Ltd, Knoxville, TN, USA). By ordered subset expectation maximization 3-dimension/maximum a posterior probability (OSEM3D/MAP) algorithm and attenuation correction information derived from the CT, the 20-min list-mode PET data were binned into a single frame and reconstructed. The final voxel size and the matrix were 0.776 mm × 0.776 mm × 0.796 mm and 128 × 128 × 159, respectively. The SUV was calculated as: [body weight (g) × tissue activity concentration (kBq/cc) / effective injected radioactivity (kBq)]. Eventually, each regional SUVmean was measured for statistical analysis.

Transmission electron microscopy (TEM)

Rats were anesthetized and intracardially perfused with 0.1 M phosphate-buffered solution (PBS) containing 4% paraformaldehyde (PFA) and 0.25% glutaraldehyde. The cerebral auditory-associated areas (amygdala [AMY], auditory cortex [ACx], medial geniculate body [MGB], inferior colliculus [IC], prefrontal cortex [PFC], hippocampus [HP], and cerebellum [CRB]) were delineated according to anatomical atlases [20]. The tissues were dissected, washed with 0.1 M PBS (pH 7.2), and then immersed in 2.5% glutaraldehyde for 2 h at 4 °C. The sample preparation of TEM was performed as previously described [21, 22]. Processed samples were observed under a JEM-2100 TEM (Philips). The number of synaptic vesicles, postsynaptic density (PSD) thickness, synaptic cleft width, and length of the synaptic active zone were measured and analyzed with ImageJ.

GABA measurement

Rats were anesthetized and intracardially perfused with ice-cold PBS to remove blood. The brains were separated from the skull and rapidly cut into 2 mm thick slices. Samples were collected and homogenized in RIPA buffer (Solarbio), and the lysates were centrifuged at 10,000 g for 5 min to separate the supernatant. The concentration of total protein was determined using the Pierce™ BCA Protein Assay Kit (Thermo). The production of GABA was measured using an ELISA Kit for Gamma-Aminobutyric Acid (GABA; Cloud-Clone Corp) following the manufacturer’s protocols.

RNA extraction and quantitative RT-PCR

Total RNA was extracted from each cerebral auditory-associated area using the RNAprep Pure Tissue kit (TIANGEN) and RNA concentration was measured by the NanoDrop spectrophotometer (Thermo). Then, cDNA was synthesized using FastKing gDNA Dispelling RT SuperMix (TIANGEN). Finally, the specific products of c-fos, early growth response gene-1 (EGR-1), and brain-derived neurotrophic factor (BDNF) were amplified by quantitative PCR using PerfectStart® Green qPCR SuperMix (TransGen Biotech) and QuantStudio™ 6 Flex Real-Time PCR System (Thermo). GAPDH was used as a normalization control. Details about the primers are summarized in Supplementary Table 1.

Immunoblotting assay

Tissues of cerebral auditory-associated areas were lysed (RIPA buffer [Solarbio] with protease and phosphatase inhibitors [CST]) and equal amounts of supernatant proteins were separated by SDS-polyacrylamide electrophoresis (PAGE), and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Next, PVDF membranes were incubated overnight at 4 °C with primary antibodies against c-fos (Proteintech, #66590-1-Ig, 1:1000) and β-III-tubulin (Biolegend, #802,001, 1:10000), followed by 1 h room temperature (RT) incubation with horseradish peroxidase-labeled secondary antibodies (ZSGB-BIO, 1:200). Labeled proteins were visualized using electrogenerated chemiluminescence (ECL) working solution (Millipore) and the density of each band was measured by ImageJ.

Immunofluorescence microscopy

After being perfused through the heart with 4% (w/v) PFA in PBS (pH 7.4), brains were immediately removed, fixed overnight in 4% PFA, and immersed in 20% (w/v) and 30% (w/v) sucrose/PFA to dehydrate until they sunk to the bottom. Coronal brain sections (15 μm thick) were obtained using a freezing microtome (Leica) through the auditory areas according to the stereotaxic atlases [20]. For immunolabelling, slices were permeabilized with 0.3% Triton X-100/PBS for 30 min at RT, blocked with 10% goat serum/PBS for 90 min at RT, and then incubated with primary antibodies anti-c-fos (CST, #2250, 1:200) overnight at 4 °C. The next day, after washing in PBS, the slices were incubated with appropriate Alexa Fluor 488-conjugated secondary antibodies (1:200, ZSGB-BIO) for 90 min at RT. Subsequently, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma) for 10 min at RT. Images were acquired on a confocal laser scanning fluorescence microscope (ZEISS). Areas covered by antibody-fluorophores and their numbers were analyzed with ImageJ.

Immunohistochemical analysis

The brain tissues were embedded in paraffin and sliced into 4-µm-thick coronal sections using a sliding microtome (Leica). After dewaxing, sections were boiled by microwave in retrieval solutions to expose antigens, followed by incubation in 3% (v/v) hydrogen peroxide (H2O2) for 30 min at RT. Then, sections were blocked with 10% (v/v) goat serum in 0.3% (v/v) Triton X-100/tris-buffered saline (TBS) for 90 min at RT and incubated with a mouse anti-c-fos antibody (Proteintech, #66590-1-Ig, 1:200) overnight at 4°C. The next day, after washing in TBS, the sections were reacted with biotinylated goat anti-mouse IgG (Vector Laboratories) for 90 min at RT. Subsequently, the slices were developed by VECTASTAIN Elite ABC-HRP kit (Vector Laboratories), and the immunohistological positive stainings were visualized by 3, 3’-diaminobenzidine (DAB) kit (Vector Laboratories) according to the manufacturer’s protocols. Finally, slices were counterstained with hematoxylin, dehydrated, and mounted. Images of brain tissue slices were captured with microscopic observation (Leica) and the quantification of c-fos positive nuclei were processed by ImageJ.

Statistical analysis

Statistical analyses were performed with SPSS 19.0 (SPSS Inc., Chicago, IL) and GraphPad Prism 6 (GraphPad Software, La Jolla, CA). All the data are presented as the mean ± standard deviation (SD). Kolmogorov-Smirnov test was used to assess data distribution and Spearman’s rank correlation test was applied to analyze the correlation of different variables. The statistical significance of differences among more than two groups was tested by one-way ANOVA. Differences within the experimental groups were determined by two-way ANOVA and Student’s t test. All tests were 2-sided, and values of P < 0.05 were considered statistically significant.