BPPV diagnosis and treatment

After a thorough physical examination and a review of clinical history, each candidate underwent the supine head-turning test or Dix-Hallpike test. In most cases (about 90%), BPPV was involved with the posterior semicircular canal, and most patients were diagnosed with canalithiasis type. Thus, only the canalithiasis type of BPPV patients involved with the posterior semicircular canal in this study. Sixty patients who were included were randomly assigned to two groups. One group was treated with Betahistine (12 mg/time, 3 times/day); the other group was treated with placebo. This treatment was continued for 4 weeks. Blood samples were then drawn at the end of 4 weeks. All patients signed an informed consent form approved by the second affiliated hospital of air force medical university Institutional Review Board prior to the first blood sampling.

Measurement index

The primary outcomes were the duration of residual dizziness and scores of the 25-item Dizziness Handicap Inventory (DHI) [17, 18] and the Berg balance scale (BBS) [19]. All patients answered the questionnaires after 4 weeks of treatment. The duration of residual dizziness was used to assess the efficacy of Betahistine treatment on improving the residual dizziness.

The DHI score was used to help patients to rate the dizziness-related physical impairments, activity limitations, and restrictions in participation [17, 18]. The DHI divided into three subdomains of impact on daily life: functional, emotional and physical (Additional file 1: Table S1). Patients were asked to complete the Dizziness Handicap Inventory (DHI) consisting of 25 questions, and a total score (0–100 points) is obtained by summing ordinal scale responses, higher scores indicating more severe handicap. The 25 items were grouped into three dimensions: functional, emotional, and physical aspects of dizziness and unsteadiness. There were nine questions within each of the functional and emotional dimensions; with a maximum score of 4 for each item. Within the physical dimension there were seven questions with a maximum score of 4 for each.

BBS is a valid tool to estimate balance condition. BBS comprises 14 balance-related tasks [19, 20]. The scale requires 15 min to complete and measures the patient's ability to maintain balance for a specified duration, either statically or while performing various functional movements. The items are scored from 0 to 4, with a score of 0 representing an inability to complete the task and a score of 4 representing independent item completion. A global score is calculated out of 56 possible points. Scores of 0 to 20 represent balance impairment, 21 to 40 represent acceptable balance, and 41 to 56 represent good balance. The BBS measures both static and dynamic aspects of balance.

Establishment and treatment of vestibular function vertigo model

Four-week-old Kunming SPF mice (25 ± 0.5 g), Slc26a4loop/loop mutant and wild-type mice (25 ± 0.5 g) were purchased from the Experimental Animal Center of the Air Force Military Medical University (Xi’an, China). The mice were kept under controlled conditions at a temperature of 22 ℃ and humidity of 70%, with a 12-h light–dark cycle, and allowed free access to food and water. All experimental animal protocols were reviewed and approved by the Ethics Committee of the second affiliated hospital of air force medical university for the use of laboratory animals.

A total of 70 mice were randomly divided into five groups, including the control group, gentamicin group, Betahistine group and Betahistine + SCH772984 group. Mice of gentamicin group were subcutaneously injected with gentamicin (150 mg/Kg); mice of Betahistine group were subcutaneously injected with gentamicin (150 mg/Kg) and Betahistine (10 mg/Kg); mice of Betahistine + SCH772984 group were subcutaneously injected with gentamicin (150 mg/Kg), Betahistine (10 mg/Kg) and SCH772984 (12 mg/Kg); mice of Betahistine + GW9662 group were subcutaneously injected with gentamicin (150 mg/Kg), Betahistine (10 mg/Kg) and GW9662 (1 mg/Kg). Mice of the control group were subcutaneously injected with normal saline according to 0.10 mL/Kg body weight. The drug was diluted with normal saline and injected for 15 days.

Establishment and treatment of BPPV model mice

A report showed that pathological findings of displaced otoliths were consistently found in the inner ear of Slc26a4loop/loop mutant mice with severe vestibular dysfunction, a mouse model with a genetic predisposition for ectopic otolithiasis with clinical relevance to BPPV, and this unique mouse model may serve as a platform for further studies of BPPV pathophysiology and for the development of novel therapeutic approaches in live animal models [21]. So, in our study, we constructed severe vestibular dysfunction Slc26a4loop/loop mutant mice as a BPPV model.

Thirty-six Slc26a4loop/loop mutant mice were randomly divided into two groups, including Slc26a4loop/loop group and Betahistine group. Mice of Betahistine group were subcutaneously injected with Betahistine (10 mg/Kg). Slc26a4loop/loop mutant mice and wild-type mice were subcutaneously injected with normal saline according to 0.10 mL/Kg body weight. The drug was diluted with normal saline and injected for 15 days.

Preparation and delivery of adenoviral vectors in vivo

Adenoviral vectors containing the enhanced green fluorescent protein gene were purchased from Life Technologies (Shanghai, China). CTRP-overexpression recombinant adenoviruses (up-Ad-CTRP, 2.0 × 1010 pfu/mL) and blank control overexpression recombinant adenoviruses (up-Ad-GFP, 1.1 × 1011 pfu/mL), interfering recombinant adenoviruses (down-Ad- CTRP, 1.1 × 1011 pfu/mL), and blank control interfering recombinant adenoviruses (down-Ad-GFP, 5 × 1010 pfu/mL) were provided by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Then, 20 μL of adenovirus solution (1 × 107 particles/μL) were injected via the intratympanic injection into designated group.

Auditory brainstem response (ABR) testing

Auditory brainstem response (ABR) was evaluated as previously described [22]. We tested only young mice (8–14 weeks old) with Slc26a4loop/loop mutant mice and wild-type mice to preclude complications from the possible age-related hearing loss. A broad-band click stimulus or pure tone pips, at 8, 16 and 32 kHz each, produced by computer (Tucker Davies) was delivered using headphones (Intelligent Hearing Systems) with an attached plastic tube to channel the sound into the ear of the mouse, which had been anaesthetized with avertin (tribromoethylic alcohol/tert-amylic alcohol) at 3.5 mg/10 g of body weight. Mice were kept warm on a heating pad during ABR recording, and tested in a chamber designed for compartmental assessment (Ugo Basile). Subdermal electrodes were inserted behind the ear pinna of the side to be recorded (active), on the top of the head (reference) and at the front of the head (ground). The unrecorded ear was plugged with modelling clay. We analyzed both ears. The ABR threshold is the lowest intensity producing a recognizable ABR pattern on the computer screen (that is, at least two peaks) using 250 stimuli. We determined thresholds for each ear. The maximum intensity of stimulation was 100 dB SPL.

Behavioral testing

To evaluate vestibular function of Slc26a4loop/loop mutant mice and wild-type mice, we utilized a battery of behavioral tests with standardized scoring systems as previously described [21]. For the swimming test, the mouse was gently inserted into a bath of warm water and watched carefully. Normal swimming performance were scored with zero, demonstrating regular performance. Mice with irregular swimming were scored with one point, while mice with immobile floating were scored with two points. Mice with an inability to swim were rescued immediately to avoid drowning and ranked with the maximum score of three points. For the trunk curl test, the mouse was gently hung from his tail 10 cm above a clean surface with a metal grid. Normal behavior was demonstrated by a reaching position, with a score of zero, by the extension of limb and head forward and downward aiming to the floor. Mice with an abnormal behavior, ranked with a score of 1, tried to climb towards the examiner s hand, curling the body upward reaching with its head to the tail. The remaining three behavioral tests were based on observation of each mouse in a clean cage. A positive circling behavior was scored with 1 point, while a head tilt gained another point. For gait observation, we defined a scale of zero (normal gait) to three points (incapacity) for mice unable to walk with a typical habitus of a tilted trunk from head to tail lying on the cage floor. All mice in this study were tested between 8 10 weeks of age. The score of each test was normalized and a total sum of the cumulative score was calculated upon completion of the behavioral pipeline, giving each test 20 percent of the final score.

Evaluation of vestibular function

The vestibular system is known as the balance organs of the inner ear, constituting three roughly orthogonal semicircular canals that sense rotational movements, as well as two otolith organs (the utricle and the saccule) that sense gravity and linear accelerations [23]. As part of the vestibular system, the otoconial membrane and the internal organic matrix of otoconia consist of glycoproteins and sulfated glycosaminoglycans complexes in the form of proteoglycans [24]. Otoconia are crucial for correct processing of orientation and positional information. Mice lacking otoconia cannot sense the direction of the gravity vector and cannot swim properly [25]. In our study, assessment of vestibular function was performed with three tests as previously reported, including the air righting reflex test, the time of contact righting reflex test and the swimming test [25,26,27].

The air righting reflex test is an antigravity reflex test in mice that depend on vestibular function [27]. Mice were supine and dropped from a height of 50 cm onto a soft filling surface. The average percentage of trials each mouse landed on all four feet was determined from five attempts for each mouse.

The time of contact righting reflex test was performed, and the mice were placed in a 60 mL opaque plastic syringe so that they could roll over but could not turn around or rear. The syringe was inverted until the animal was in the supine position, and the average time for each mouse to recover the upright position within the syringe in three trials was determined.

In the swimming test, the mice were placed in a 20 × 40 cm plexiglass cage filled with water at room temperature for 60 s. In three trials in each mouse, the average swimming time with the head above water was measured.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA isolation was performed using the Trizol reagent (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. According to gene sequences published in GenBank’s database, primers were designed using Primer 5.0 design software and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Reverse transcription was performed with approximately 0.5 μg of total RNA into cDNA with the Superscript™ reverse transcription system (Takara, Dalian, Japan). Aliquots of cDNA were used for PCR amplification with primer-specific. RT-qPCR reaction was performed as follows: 95 ℃ for 3 min, 35 cycles of denaturation at 95 ℃ for 30 s, 58 ℃ for 45 s, and 72 ℃ for 30 s and 8 min at 72 ℃ using SYBR Green PCR master mix reagents (Takara, Dalian, Japan) in the ABI StepOne Plus Real-time PCR system. The relative quantification of mRNA expression levels was calculated using the 2 − ΔΔCT method.

Western blotting

Total protein was extracted from the inner ear tissues by using ice-cold RIPA buffer (Sigma, St. Louis, MO, USA). Proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to PVDF membranes (Sigma, St. Louis, MO, USA). The PVDF membranes were blocked with 5% skim milk at room temperature for 2 h. Then, the membranes were placed into the primary antibody solution for incubation at 4 ℃ overnight. The membranes were washed 3 times/5 min with TBST and placed into the secondary antibody solution for incubation at room temperature for 1 h and rewashed 3 times/5 min with TBST. The band development of protein was visualized by using the ECL chemiluminescence reagent at a chemiluminescent detection system, and the grey values of target proteins were performed image analysis by using Image J software. Antibodies used in this study are as follows: CTRP1 (1:600), CTRP3 (1:600), CTRP6 (1:600), CTRP9 (1:600), CTRP12 (1:600), ERK1/2 (1:1000), AKT (1:1000), PPARγ (1:1000) and β-actin (1:1000). All primary antibodies were purchased from Abcam, Cambridge, MA, USA.

ELISA assay

Blood samples were centrifuged at 4 ℃ for 15 min at 3600 rpm to separate serum and analyzed CTRP1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15 and adiponectin levels in serum with the ELISA kit, according to the manufacturer’s instructions. All ELISA kits were purchased from AdipoGen Life Sciences.

Statistical analysis

All data are shown as the mean ± SEM of three or more independent experiments were performed. Statistical differences were evaluated by using a one-way analysis of variance (ANOVA) and the Student’s t-test. In the univariate analysis, comparisons of numerical parameters between both groups were evaluated by using the Student’s t-test, and multigroup comparisons of the means were evaluated by using a one-way ANOVA test. Values were considered statistically significant differences at P < 0.05. Statistics were performed with SPSS Statistics version 20.0 software.